Background
Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin networks and link them to cellular membranes during cell movements [
1]. Filamins crosslink cortical filamentous actin into a dynamic orthogonal network and thereby confer membrane integrity and protection against mechanical stress. In addition to actin, filamins bind to numerous other proteins such as transmembrane receptors and signaling molecules and provide scaffolding functions and regulate multiple cellular behaviors [
2]. Although filamins are classically known as cytoplasmic structural proteins, recent studies suggest that filamins are emerged as essential scaffolding proteins that play roles in cell signaling [
2]. In addition, filamins interact with transcriptional factors to regulate their function and become members of transcriptional complex in the nucleus [
2]. There are three members of the filamin family, filamin A (FLNA), filamin B (FLNB) and filamin C (FLNC). Both human
FLNA and mouse filamin A (
Flna) genes are located on the X chromosome. During embryogenesis as well as in adults, FLNA is the most abundant isoform, is ubiquitously expressed throughout the body and appears to be the major filamin responsible for cardiovascular development.
Many studies have reported increased expression of FLNA in human cancer tissues such as hepatic [
3], breast, and astrocytoma [
4] as well as in different cancer cell lines and human lung cells [
5]. FLNA may mediate the effects of signaling pathways on both cancer and endothelial cell motility during tumorigenesis. In addition, the RAS-signaling pathway has attracted considerable attention as a target for anticancer therapy because of its important role in carcinogenesis [
6]. Interestingly, in mammalian cells, the generation of actin-based dynamic motile structures is regulated by small GTPases of the Rho family and FLNA interacts with these GTPases [
7]. Following integrin binding to extracellular matrix ligands, small GTPases are activated, leading to actin polymerization and the formation of lamellipodia and filopodia. Branched actin networks are particularly important for the formation of lamellipodia that are believed to be the actual motors that pull cells forward. Filopodia originate from the pre-existing lamellipodial actin network that is prevented from capping and, as a result, can elongate at the leading edge of the lamellipodia. Mutations in the K-RAS gene render the protein unable to hydrolyze GTP and have been found in 20–30% of non-small-cell lung cancers [
8]. The small GTP-binding proteins K-RAS, H-RAS and N-RAS belong to a family of oncoproteins associated with many types of other human cancer. The
K-RAS gene is designated
Kras2 in the mouse. RAS proteins interact with a number of effector proteins that in turn activate important signaling pathways, including the RAF/MEK/ERK and the PI3K/PKB/AKT pathways [
8]. The complexity of the RAS signaling pathway and the difficulty of targeting the RAS protein itself necessitate continuous searches for additional mechanisms that regulate RAS-induced tumor growth.
A recent study showed that an interaction between active RAS and FLNA is responsible for maintaining endothelial barrier function [
9]. Loss of the RAS-FLNA interaction promotes VE-Cadherin phosphorylation and changes in downstream effectors that lead to endothelial leakiness. Interestingly, complete
Flna deficiency results in embryonic lethality in mice due to severe cardiac structural malformations [
10]. In addition, it has been reported that breakdown of the endothelial lining could weaken the blood vasculature, leading to vascular abnormalities [
10].
Despite the many studies focusing on the expression and function of FLNA in tumor cells, its role in endothelial cells and cell migration, very little is known about the importance of FLNA in endogenous tumor growth. In addition, the specific role of FLNA in oncogenic angiogenesis has not yet been explored. In this study, we used two different tumor models in mice to determine the role of FLNA in K-RAS–induced lung tumor formation and the role of endothelial FLNA during tumor growth.
Discussions
In this study, we demonstrated that knockout of Flna reduces K-RAS–induced lung tumor development in vivo and reduces the proliferation of K-RASG12D–expressing fibroblasts in vitro. The reduced fibroblast proliferation was associated with reduced levels of activated ERK and AKT. Moreover, we showed that targeting Flna specifically to endothelial cells reduces their migratory ability and retards local tumor growth.
Endogenous activation of K-RAS
G12D in the lung results in adenocarcinoma that originates in terminal and respiratory bronchi or in the alveolar epithelium [
12]. Adenocarcinoma of the lung is the most common type of lung cancer in lifelong non-smokers [
19]. Studies of human lung cancer have shown that adenocarcinoma is the only subtype associated with RAS mutations [
20]. In the
Kras2LSL model, inhalation of
Cre-adenoviral vector results primarily in infection of respiratory epithelial cells, where Cre deletes the “floxed” stop cassette to activate the expression of K-RAS
G12D from the endogenous promoter. In the
Flnao/flKras2LSL/+ mice, Cre expression simultaneously inactivated
Flna. Although
Flna deficiency significantly reduced lung tumor development, it did not abolish tumors. This could conceivably be caused by partial recombination, where the K-RAS allele would be activated by Cre but
Flna would not be inactivated in every cell as reported [
12,
19]. We believe that Cre-adenovirus infection in the
Kras2
LSL
lung does not result in Cre expression and recombination of “floxed” alleles in endothelial cells as these cells are located at some distance from the respiratory epithelium.
The RAS/RAF/MEK/ERK and RAS/PI3K/PTEN/AKT signaling pathways are cascades regulated by phosphorylation and dephosphorylation by specific kinases, phosphatases, as well as GTP/GDP exchange proteins, adaptor proteins and scaffolding proteins [
21]. These pathways play key roles in the proliferation of tumor cells and growth of tumors. Therefore, inhibitors targeting these pathways have many potential uses for suppression of cancer. However, cancer therapy is often complex as there are relatively few cancers which proliferate in response to a single molecule interaction which prevents them from being treated with a monospecific drug. Therefore, new targets need to be identified to develop more effective treatments. In this study, we showed that cellular K-RAS–induced proliferation was reduced by
Flna deficiency. The effect was associated with impaired activation of the RAS downstream effectors ERK and AKT. This finding suggests that targeting FLNA might be considered in combined treatment with established targets. The impact of
Flna deficiency on cellular proliferation has not been reported in normal MEFs [
10], however, we observed that
Flna-deficiency impairs proliferation of these cells when induced by K-RAS.
What is the mechanism behind the reduced tumor growth and proliferation in K-RAS–expressing cells? One potential explanation is that FLNA acts as a scaffolding protein and is required for efficient spatial and temporal activation of effectors in the RAS pathway and that the absence of FLNA directly affects RAS signaling. Another is that FLNA is involved in regulating the dynamics of the actin cytoskeleton and that the impact of
Flna deficiency on tumor growth reflects a more general role for the protein in cellular structure and function. A third, perhaps more intriguing possibility, is that the FLNA protein can be cleaved in the hinge region and regulate gene transcription in the nucleus [
22]. Over the next few years, studies will likely shed light on these different possibilities.
Because FLNA has been shown to be important in vascular cells [
10,
23], we were interested in defining the impact of
Flna deficiency on both normal and tumor endothelial cells. Interestingly, mice lacking
Flna in endothelial cells had no apparent phenotypes. Cardiac development and function appeared to be normal and vascular integrity was unaffected. This finding was surprising for several reasons. First,
Flna-deficient mice showed prominent cardiovascular abnormalities as well as extensive defects in cell–cell junctions that were particularly prominent in vascular endothelial cells [
10]. And second, multiple functions of FLNA in endothelial cells have recently been reported including caveolae internalization and trafficking [
24] and chemotaxis [
25]. Interestingly, a crucial role for another filamin, FLNB, in endothelial cell migration and in the angiogenic process in adult endothelial cells has been reported [
26]. As both filamin genes are highly conserved and the filamin proteins exhibit high amino acid identity and can also form heterodimers [
2], it is likely that FLNA and FLNB have both unique and overlapping roles in the vascular endothelium. Regardless, our findings suggest that FLNA might not be as important for endothelial cell function as had previously been appreciated. We did, however, observe reduced migration of
Flna-deficient endothelial cells. Moreover, we found that fibrosarcoma and melanoma tumor growth under the skin of mice lacking
Flna in endothelial cells was reduced. As FLNA was specifically deficient in vascular endothelial cells, we observed a significantly reduced number of vascular endothelial cells, but not pericytes. This result suggests that endothelial FLNA may be important in tumor angiogenesis.
In summary, this study provides new insight into the biology of FLNA and suggests that in addition to its classically known cytoskeletal function, the protein also plays an important role in the activation of ERK and AKT signaling pathways during K-RAS–induced transformation. Additionally, mice lacking Flna in endothelial cells developed smaller tumors. Finally, the experimental approach described here should be useful for dissecting the in vivo importance of Flna in other cancers and in tumor and physiological angiogenesis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RKN, MOB, LMA designed research; RKN, MXI, SB, SSN, BR, YZ performed research; RKN, MXI, AXZ, SB, DP, JB, YC, MOB, LMA analysed the data; RKN, MOB, LMA wrote the paper. All authors read and approved the final manuscript.