Background
FGD4, a guanine nucleotide exchange factor (GEF) specific for CDC42 Rho GTPase, is an F-actin binding protein [
1] which is essential for maintenance of myelination in Schwann cells [
2]. The FGD4 gene belongs to a family of GEFs which includes FGD1, FGD2, FGD3, FGD5, FGD6 and FRG. Each contain an FYVE domain in addition to a Dbl homology (DH) domain for catalytic activity and two pleckstrin homology (PH) domains for targeting proteins to the membrane through phosphoinositide binding [
3‐
5]. The PH1 domain helps the DH domain with its catalytic activity through binding of nucleotide free small G-proteins [
6,
7]. The FYVE finger domain is responsible for membrane trafficking and phosphoinositide metabolism through interaction with phosphoinositol-3-phosphate (PI3P) [
8]. All members of the FGD family show significant sequence homology within DH, PH and FYVE domains, but FGD4 has an additional actin binding FAB domain at the 5′ end which is not present in other FGD proteins [
9]. The DH domain and the PH1 domains are responsible for activation of CDC42 through GTP exchange for GDP, whereas the FYVE domain and the PH2 domains are involved in indirect activation of Rac [
10]. The functions of FGD4 are multifaceted and includes bundling of F-actin through F-actin binding with FAB domains, activation of Rho GTPase signaling pathway through increased concentration of GTP bound CDC42, and plasma membrane association through FYVE domain binding with phosphoinositides and PI3P. FGD4 domain structures suggest its role as a cross linker between membrane structure and actin cytoskeleton. Loss of function mutations in the FGD4 coding sequence leading to expression of truncated FGD4 causes motor-sensory neuropathies or autosomal recessive Charcot-Marie-Tooth (CMT) disease type 4 [
2,
11,
12]. The effect of mutation is mediated through inhibition of guanine nucleotide exchange function leading to reduced CDC42 activity, which results in demyelination of peripheral nerves [
13].
RhoGEFs are known to be involved in cancer progression through activation of G-proteins, including members of Rho GTPases. A number of RhoGEFs are overexpressed in prostate cancer, including Rac1 GEF P-Rex [
14] and Vav3 GEF for RhoA, Rac and CDC42 [
15,
16], and are responsible for promoting metastasis and castration resistant prostate cancer (CRPC) by regulating androgen receptor (AR) activity. Activated CDC42 promotes cell proliferation through its interaction with the effector proteins MLK [
17] and ACK1/TNK, which activates ERK and AKT. Additionally, activated CDC42 modulates cell migration through interaction with PAK4 [
18] and MRCK [
19], activation of LIMK 1/2 and reorganization of actin cytoskeleton [
20]. Although the function of CDC42 is well documented, the involvement of CDC42 specific GEF FGD4 in prostate cancer has not been reported.
Prostate cancer accounts for one of the leading causes of cancer related deaths in the Western world [
21]. Advances in detection methods including magnetic resonance imaging and prostate specific antigen (PSA) screening [
22,
23] helped detection of prostate cancer at earlier stages and designing effective therapeutic strategies, such as surgical or radiation therapies or in some cases active surveillance [
24,
25]. Although initially indolent and responsive to definitive treatments prostate cancer can become aggressive and present biochemical recurrence with elevated levels of PSA [
26,
27]. For patients who experience dissemination of prostate cancer after localized therapy androgen deprivation therapy (ADT) is considered as the standard of care as monotherapy or in combination therapy with other agents [
28,
29]. Nonetheless, resistance to ADT is common, which leads to development of CRPC [
30,
31]. Our earlier studies showed an association between ADT, using AR antagonist Casodex, and upregulation of FGD4 in androgen sensitive prostate cancer cells [
32]. In this study, we show a positive correlation of FGD4 expression with aggressiveness of prostate cancer and tumor promoting properties of FGD4 in prostate cancer.
Methods
Tissue microarray and immunohistochemistry
A prostate cancer tissue microarray block (TMAs) was constructed in the Tissue Core Facility at the Moffitt Cancer Center. The TMA contains 0.5 cm cores of de-identified tissues from 263 patients. The composition of tumor samples is presented in Table
1. The H&E slides of each sample was reviewed and the diagnosis confirmed by a pathologist (DC), before being included in the TMA. The area of interest was outlined by the pathologist for sampling and the TMA was constructed using a TMA Instrument (Tissue Arrayer, Beecher Instruments). A TMA section 3 μm in thickness was stained using an anti-FGD4 antibody (GeneTex NIN3). Dual staining of TMA was performed using the following protocol. Deparaffinized TMA was subjected to antigen retrieval in citrate buffer (pH 6.0) in a pressure cooker for 45 mins. Next, the TMA was blocked by peroxidase (3%) blocking solution followed by goat serum. Primary antibody was applied and incubated at room temperature for 1 h. Anti-rabbit secondary antibodies (Dako’s Envision anti rabbit RTU polymer system) were applied next and incubated at room temperature. Next, ImmPACT™ DAB substrate (Vector Laboratories) was added, incubated at room temperature, washed, and counterstained with SelecTec hematoxylene (Leica). The stained TMA was imaged using Aperio AT2 digital whole slide scanner (Leica) and evaluated using ImageScope image analysis software. The staining intensity analysis and the scoring was also done by a pathologist. The immunostaining was scored using the Allred scoring system [
33].
Table 1
Compostition of the prostate TMA
BPH | 23 | GS7 | 39 |
PIN-Lo | 24 | GS 8, 9, 10 | 51 |
PIN-Hi | 15 | M | 10 |
GS6 | 32 | AI (GS 7–10) | 13 |
Cell lines and transfection
PC-3 cells obtained from ATCC were cultured in F12 HAM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Invitrogen). The androgen dependent LNCaP-104S cells (a gift from Dr. Shutsung Liao, University of Chicago) were isolated from the parental LNCaP cells and characterized [
34]. LNCaP-104S cells were maintained in DMEM containing 10% FBS, 1 nM DHT (Sigma-Aldrich) and 1% antibiotic/antimycotic. C4-2B cells are developed from vertebral metastasis of LNCaP xenografts [
35] (a gift from Dr. Leland Chung, Cedars-Sinai Medical Center). C4-2B cells were maintained in RPMI with 10% FBS and 1% antibiotic/antimycotic. All cells are rigorously monitored for mycoplasma contamination using DAPI staining method and authenticated through STR profiling (Additional file
1: Table S1). For inhibition of FGD4 expression, 4 FGD4 siRNAs (FlexiTube Gene Solution, Qiagen) were used for transient transfection using Lipofectamine RNAiMax (ThermoFisher) transfection reagent. Three control siRNAs were used for transient transfection in parallel as the controls. Transfected cells were harvested at 48 h for subsequent experiments. For ectopic expression, DNA of full length coding sequence of
FGD4 gene was synthesized (GenScript), cloned into pcDNA 3.1 and pcDNA 3.1-EGFP mammalian expression vectors (FGD4 pcDNA and pcDNA 3.1 FGD4-EGFP) and sequence verified. FGD4 pcDNA, pcDNA 3.1 FGD4-EGFP, pcDNA 3.1 MECP2-EGFP, or the empty vector as the control, was used for transient transfection using Lipofectamine (Invitrogen). Cells were used after 48 h for subsequent experiments.
RNA extraction and quantitative real-time PCR
Total RNA from transfected cells was extracted using RNeasy kit (Qiagen). Total RNA was converted to cDNAs using QuantiScript Reverse Transcriptase (Qiagen) and used for quantitative PCR using FGD4 QuantiTect forward and reverse primers (Hs_FGD4_1_SG QuantiTect, Qiagen). The primers were designed to provide maximum efficiency for relative quantification. Quantitative PCR was conducted using Rotor-Gene SYBR Green PCR reagents (Qiagen) and Qiagen Rotor-Gene Q thermal cycler and data analyzed using Rotor-Gene-Q software. DNA concentration was assessed using SYBR Green fluorescence and Ct values generated were normalized using Ct values of RPL13A and GAPDH normalizer genes. The Ct values were used to derive ΔΔCt values using the miRNome analysis software (System Biosciences).
Western blotting
Lysates of transfected PC-3, LNCaP-104S and C4-2B cells were prepared and used for immunoblotting using anti-FGD4, anti-E-cadherin, anti-SLUG, anti-phospho PAK, anti-phospho cofilin, anti-GAPDH and anti-alpha-tubulin antibodies (Additional file
1: Table S2). Signals were detected using enhanced chemiluminescence (ECL) detection method. GAPDH and alpha-tubulin were used as the loading controls. Comparative analysis of the target protein expression was performed using densitometric analysis of the normalized peptide band intensity.
Table 2
p Values pairwise comparison of FGD4 expression between tumor categories
BPH | 0.09296 | 0.08364 | 0.00244 | 0.00544 | 0.00544 | 0.00138 | 0.00094 |
PIN-Lo | | 1.0 | 0.72634 | 0.68916 | 0.41222 | 0.09296 | 0.0601 |
PIN-Hi | | | 0.70394 | 0.42952 | 0.79486 | 0.12852 | 0.03156 |
G6 | | | | 0.50286 | 0.72786 | 0.04884 | 0.01078 |
G7(3 + 4) | | | | | 0.71138 | 0.09296 | 0.06724 |
G7(4 + 3) | | | | | | 0.05238 | 0.07346 |
G8910 | | | | | | | 0.96012 |
Cell proliferation and drug sensitivity assays
PC-3 and LNCaP-104S cells were seeded in 96 well plates and transfected with FGD4 siRNAs or control siRNAs after 24 h or 48 h after seeding. Transfection medium was replaced with fresh medium after 8 h of transfection. For drug sensitivity assays, the media were replaced with 10 μM Casodex or DMSO in 20% charcoal-stripped FBS (CS-FBS) containing growth medium (LNCaP-104S) or 5 nM and 25 nM Docetaxel, or the vehicle in regular complete growth medium (PC-3). Cell proliferation was detected at 48 h after transfection using MTS based Cell Titer Aqueous One Solution cell proliferation assay kit (Promega).
Flow cytometry
PC-3 and LNCaP-104S cells were seeded in a 12-well dish and transfected with FGD4 siRNAs or control siRNAs after 24 h or 48 h. Cells were harvested at 48 h post transfection and resuspended in cold PBS before being placed on ice. Ice-cold methanol was added to fix and permeabilize the cells. The cells were left at -20 °C in methanol for 30 min. The tubes were returned to ice and cold PBS was added to the tubes. Cells were incubated on ice for an additional 5 min, centrifuged and rinsed with PBS twice and resuspended in PBS containing 50 μg/mL RNase and 2% Bovine Serum Albumin (BSA) in PBS. The tubes were incubated for 15 min at room temperature and then diluted with 2% BSA in PBS. Propidium iodide (PI) in 2% BSA in PBS solution was added to each tube to achieve 50 μg/ml and the tubes were incubated in the dark at room temperature for 1 h. The cells were run in a BD Accuri flow cytometer until a count of 10,000 PI-stained events was obtained per sample. FlowJo Analysis software was used for cell cycle analysis.
Scratch assay
PC-3 and C4-2B cells were seeded into 24-well plate in growth medium containing 5% FBS and transfected with FGD4 siRNAs or control siRNAs, or FGD4 pcDNA or the control vector after 24 h of seeding. Transfection media was replaced with fresh media after 8 h and incubation continued for 48 h. Wounds in the form of a straight scratch were created using a micro pipet tip. Following the scratch, cells were rinsed twice with PBS and incubated in media containing 5% FBS at 37 °C. Migration within the scratch was imaged at 0 h and 14 h or 24 h using a Nikon eclipse TE200 inverted microscope coupled with Nikon elements F 2.20 software. The average width of the scratch was determined using Image J software. The relative rate of migration was determined through the analysis of the remaining width of the scratch at 14 h and subtracting that from the 0 h. Next, the ratio of the distance traversed by the FGD4 DNA or FGD4 siRNA transfected and control DNA or control siRNA transfected cells respectively was determined.
CDC42 activation assay
PC-3 cells were seeded and grown to 70% confluence before being transfected with FGD4 siRNA or control siRNA. Cells were harvested at 48 h after transfection and subjected to CDC42 activation assay using CDC42 G-LISA Activation Assay kit (Cytoskeleton) according to the manufacturer’s protocol. Briefly, cell lysates were prepared and equal amounts of protein were added to the tubes containing GTP-binding protein and incubated with shaking at 4 °C. Unbound proteins were washed off and antigen presenting buffer was added in tubes and incubated at room temperature. Primary antibodies were added and incubated at room temperature. Next, tubes were washed and secondary antibodies were added to them. Samples were incubated at room temperature, washed and HRP detection reagent mix followed by HRP stop buffer were added and absorbance of the developed color was detected in a spectrophotometer at 490 nm.
Dual label immunofluorescence
C4-2B cells were seeded in Permanox Lab-Tek chamber slides, and after 24 h transfected with either a FGD4-EGFP fusion construct, pcDNA-FGD4–3’EGFP, or a control fusion protein, pcDNA-MECP2-EGFP. After 24 h cells were treated with DMSO or ROCK inhibitor Y-27632 (20 μM) (Selleck Chemicals) or kept untreated. Cells were incubated for 24 h and fixed with 4% paraformaldehyde in PBS and permeabilized with 0.01% Triton-X-100. To visualize the actin cytoskeleton, cells were stained with 0.26 μM Alexa Fluor 647-phalloidin for 10 min (Cell Signaling). Cells were mounted with DAPI Fluoromount-G (Southern Biotech) and imaged using a Leica SP5 confocal microscope and images were analyzed using Leica LAS AF software suite.
Statistical analysis
All statistical analyses were performed using Student t-test or one-way ANOVA for independent measures using GraphPad Prizm.
Discussion
In this study, we showed that FGD4 expression levels are increased in advanced prostate cancer compared to the luminal cells in benign prostatic hyperplasia. Additionally, we identified an important role of FGD4 in cell proliferation, cell cycle progression and cell migration, which is most likely mediated through activation of CDC42. We also showed the beneficial effect of inhibiting FGD4 expression on drug sensitivity.
Previous studies on FGD4 were mostly focused on its mutations and its association with demyelinating neuropathy in patients with CMT disease. A number of mutations resulting in premature termination of the protein and generation of a new donor splice site leading to truncated exon 7 and a frame shift have been detected in these patients [
13]. A recent study showed that loss of FGD4 in Schwann cells reduced endocytosis through altered recruitment of activated CDC42 to the membrane, resulting in irregular accumulation of proteins at the plasma membrane and altered myelin membrane dynamics [
2]. Nonetheless, limited information is available on the association of FGD4 with cancer. One study showed that the latent membrane protein (LMP) 1 of Epstein-Barr virus promotes increased motility of nasopharyngeal carcinoma (NPC) cells through increased CDC42 activity and remodeling of the actin cytoskeleton. The effect of LMP-induced increased motility is mediated through its direct interaction with FGD4, leading to enhanced GEF activity and thereby increasing activation of CDC42 [
44]. Metastasis suppressor SSeCKs (AKAP12) regulates chemotaxis through modulating localization of chemotaxis regulators including Rac1 and CDC42. Loss of SSeCKs enriches membrane localization of FGD4 through its interaction with PIP2/3 via its PH and FYVE domains and with the actin filament via its FAB domain, which leads to localized activation of CDC42 [
45].
In our earlier studies, we reported up-regulation of FGD4 expression in Casodex resistant prostate cancer cells, which is possibly the result of loss of expression of miR-17-92a cluster miRNAs in these cells. We further showed that FGD4 destabilization by miR-17-92a was through binding at the 3’UTR [
36]. In this study, we provide evidence for the first time that FGD4 expression is upregulated in advanced prostate cancer with higher Gleason Scores and CRPC status. Our observation is supported by the TCGA database analysis showing FGD4 DNA copy number amplification and mRNA upregulation in a subset of neuroendocrine prostate cancer and in prostate carcinoma. Our results add another GEF in the growing list of GEFs that play important roles in prostate cancer and regulation of AR activity. One such GEF is VAV3, which is overexpressed in androgen independent prostate cancer cells and tissues, and up-regulates AR activity in prostate cancer cells [
46].
Our results showed the dependence of prostate cancer cells on FGD4 expression for normal functioning, as down regulation of FGD4 reduced cell viability and led to G2/M arrest. The role of activated CDC42 in regulation of metaphase is well documented, specifically during spindle orientation [
47], maintenance of centrosome integrity [
48] and bi-orient attachment of spindle microtubules to kinetochores [
49]. CDC42 is the primary Rho GTPase that participates in the chromosome alignment at the metaphase plate. Specific GEFs are identified that activate CDC42 during specific sub-stages of prometaphase/metaphase, which allows localized increase in the CDC42-GTP pool for activation of downstream effectors. Our results showing G2/M arrest upon FGD4 siRNA treatment suggests the requirement of FGD4, possibly as a CDC42 activator for progression of mitotic phases. However, the role of FGD4 in actin cytoskeleton reorganization through direct binding to actin filament [
5] cannot be ruled out as modulation of dynamics of the cortical actin is essential during spindle orientation [
50].
Another hallmark of cancer progression is enhanced cell migration. Being an actin binding protein and an activator of CDC42, FGD4 is expected to contribute in the regulatory mechanism of cell motility. In support of that, we noted a significant decrease (50%) in cell migration in scratch assays and a reduced activation of CDC42 upon inhibition of FGD4 expression in prostate cancer cells. This experiment was conducted without any chemotactic stimulus, which suggests that the inherent highly migratory nature of PC-3 cells requires an optimum level of FGD4. This assumption is further supported by our scratch assay results obtained upon ectopic expression of FGD4, which showed a significant increase (~ 55%) in cell migration. This observation is further supported by increased expression of mesenchymal marker SLUG and decreased expression of the epithelial marker E-cadherin in these cells, which suggests an indirect activation of EMT upon FGD4 overexpression. Furthermore, the function of FGD4 on modulating actin dynamics also plays an important role in promoting migratory behavior, which is through activation of CDC42/PAK/LIMK1 pathway as noted by increased activation of PAK and LIMK1 through increased phosphorylation of PAK and cofilin, a substrate of LIMK1. Activation of the CDC42/PAK pathway was further supported by our immunofluorescence analysis showing F-actin accumulation and filopodia formation at the cell periphery, which is not mediated by Rho/ROCK. Taken together, these observations establish FGD4 as a promoter of migratory behavior of advanced prostate cancer cells.
Our observations on the involvement of FGD4 in maintaining functional homeostasis of prostate cancer cells suggest its potential for being a suitable therapeutic target. Considering FGD4’s role in myelination, the possibility exists that general inhibition of FGD4 expression may lead to neuropathic effects. However, patients with CMT Disease Type 4H, in which FGD4 is absent or truncated, have very slow progression of peripheral nerve demyelination [
2]. If used in treatment of prostate cancer, inhibition of FGD4 would be limited to short-term, localized therapy, compared to the sustained inactivation of FGD4 in Schwann cells of patients with CMT Type 4H, and is unlikely to have the same effects. FGD4 has been proposed as a paclitaxel-sensitizer gene, as silencing of FGD4 improved paclitaxel sensitivity of H1155 non-small cell carcinoma cells but this effect was not observed in any other cancer cell line [
51]. Nonetheless, our results show an increased sensitivity of PC-3 cells to docetaxel and of androgen-sensitive LNCaP-104S cells to Casodex upon silencing of FGD4. It can be speculated that the effect of the microtubule stabilizing drug docetaxel on the mitotic arrest and induction of apoptosis could be aided by the interference in the G2/M progression upon silencing of FGD4. The effect of Casodex, on the other hand, has been shown to be mediated by inhibition of G1 phase progression through inactivating AR transcriptional activation [
52,
53] and induction of apoptosis through caspase dependent and independent pathways [
54]. It is possible that silencing of FGD4 can also have a synergistic effect on the Casodex mediated reduction in cell viability through its effect on G2/M arrest. However, the exact mechanistic role of FGD4 in cell cycle progression needs further study.