Raf kinase inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melanoma

Keywords: RKIP; MDA-9/syntenin, melanoma; c-Src; FAK

Cell lines and culture conditions

A clone of normal immortal human melanocytes FM516-SV (referred to as FM-516, was initially provided by Dr. L. Diamond, Wistar Institute, Philadelphia, PA), radial growth phase melanoma WM35 (obtained from Dr. Meenhard Herlyn, Wistar Institute, Philadelphia, PA), vertical growth phase melanoma WM278 and metastatic melanoma FO-1, C8161.9, SK-Mel-28 and MeWo cell lines were cultured as described previously (8). WM278 and SK-Mel-28 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA), MeWo were obtained from Dr. Robert Kerbel, Sunnybrook Cancer Center, Toronto, Canada, FO-1 were provided by Dr. Eliezer Huberman, Argonne National Laboratories, Argonne, IL, and C8161.9 was a gift from Dr. Danny R. Welch, Kansas University Medical Center, KS. All the cells were routinely checked for morphology, in vitro phenotypes (such as invasive and anchorage independent growth) and mycoplasma contamination using a PCR-based mycoplasma detection kit (Applied Biosystems, Foster City, CA).

Plasmid construction and adenoviruses

The mammalian expression plasmid CMV-HA-RKIP with a HA-tag in the amino terminus has been described previously (28, 35). Deletion mutants of pCMV5-HA-RKIP for expression in mammalian cells were generated by PCR (35) and provided by Kam Yeung, University of Toledo, Toledo, OH. The RKIP promoter has 4 E-box consensus sites (E-box: CANNTG) clustered in two locations in the proximal RKIP promoter. The RKIP promoter driven luciferase reporter plasmid (CMV) contains all four E-box-binding sites and was used for our studies. The NF-κB promoter luciferase reporter construct contains three NF-κB consensus-binding sites upstream of the luciferase gene. The genomic sequence of MDA-9 was amplified by PCR using genomic DNA as template and primers, sense: 5′-CTGCAAAAATGTCTCTCTATCC-3′ and anti-sense: 5′ GGTGCCGTGAATTTTAAACCTCAG-3′. The PCR product was cloned into pREP4 expression vectors from where it was digested and released with Xho and BamH1 and subcloned into the pcDNA3.1 (+hygro) (Invitrogen, Carlsbad, CA). Small hairpin RNA for MDA-9 was constructed with pSilencerTM hygro Expression vectors according to the manufacturers protocol (Ambion Inc. TX). Specific hairpin siRNA oligonucleotides (sense 5′-GATCCGCGGATGGCACCAAGCATTTTCAAGAGAAATGCTTGGTGC CATCCGCTTTTTTGGAAA-3′ and antisense 5′-AGCTTTTCCAAAAAAGCG GATGGCACCAAGCATTTCTCTTGAAAATGCTTGGTGCCATCCGCG-3′) were annealed and ligated to pSilencer vector by T4 DNA ligase. The insertion was confirmed by sequencing and qPCR and Western blotting techniques confirmed the authenticity of the construct to down regulate MDA-9. Construction of Ad.5/3-null and Ad.5/3-mda-9, replication-incompetent serotype 5/3 Ad lacking a transgene or expressing mda-9, respectively, were prepared as described (36).

Engineering of RKIP mutant constructs

Different amino-acid substitutions within the RAF-1 binding site of RKIP, were generated by the QuickChange site-directed mutagenesis kit (Stratagene, Santa Clara, CA) according to the manufacturer’s instructions. HA-RKIP was used as template to generate the site-directed mutants.

RNA isolation and qPCR

Total RNA was extracted using QIAGEN miRNeasy Mini Kit (QIAGEN, Valencia, CA). qPCR was performed using ABI 7900 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

Western blotting and co-immunoprecipitations

Preparation of whole-cell lysates, co-immunoprecipitations and Western blotting were performed as described previously (8, 13, 14). The primary antibodies included: MDA-9 (Abnova, Walnut, CA), anti-c-Src pY418 and anti-FAK pY397 (Biosource International, Wilmington, NC), anti-c-Src, (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FAK (Transduction Laboratories, San Jose, CA), anti-RKIP (Cell signaling Inc. Boston, MA), and anti-HA (Sigma, St. Louis., MO). Blots were stripped and normalized by re-probing with anti–EF-1α antibody (Upstate Biotechnology, Waltham, MA).

Tissue microarray and immunohistochemistry

The malignant melanoma tissue microarray (IMH-369) was purchased from Imgenex Corp (San Diego, CA), and immunostaining was performed as described previously (8).

Anchorage-independent growth

Anchorage-independent growth assays were carried out as previously described (8).

In vitro Cell Invasion

Cell invasion was determined as described previously (8) in a modified Boyden chamber (BD Bioscience, Bedford, MA, USA) according to the manufacture's instructions.

Chicken embryo chorioallantoic membrane (CAM) assay for chorioallantoic membrane spontaneous lung metastases

To detect spontaneous lung metastases, we performed CAM assays as described (37, 38). Fertilized white leghorn eggs (Charles River, North Franklin, CT) were incubated in a humidified rotary incubator at 37°C for 9 days. On the 3rd day of incubation, 3 ml of albumin was withdrawn by aseptically drilling an opening in the eggshell. On day 9, single-cell suspensions of tumor cells were prepared at 1 × 10⁵ cells in 25 µL of serum-free DMEM and were inoculated within 1 to 2 h on the dropped CAM. The embryos were incubated for 8 additional days in a humidified stationary incubator at 37°C. At the end point of the experiment, the upper CAM with the developing tumor, the portions of the lower CAM, and the lungs were excised from the individual embryos and snap-frozen on dry ice for performing real-time Alu PCR analyses. Briefly, genomic DNA was extracted from harvested tissues using the Puregene DNA purification kit (Genta Systems, Minneapolis, MN). Human Alu sequences were amplified by RT-PCR using sense, 5'ACGCCTGTAATCCCAGCACTT-3' and antisense 5'-TCGCCCAGGCTGGGTGCA-3') primers. PCR conditions included polymerase activation at 95°C for 4 min followed by 30 cycles at 95°C for 30 sec, 63°C for 30 sec, and 72°C for 30 sec.

Statistical analysis

The data are reported as the mean ± S.D. of the values from three independent determinations and statistical analysis was performed using the Student's t test in comparison with corresponding controls. Probability values < 0.05 were considered statistically significant.

RKIP expression is downregulated in metastatic melanoma cells and inversely correlates with MDA-9 expression

The level of RKIP mRNA and protein is significantly lower, whereas the level of MDA-9 is higher, in four progressed metastatic melanoma cell lines in comparison with FM-516 SV40 T/t Ag-immortalized human melanocyte cells (indicated as FM-516) and WM-35 radial growth phase primary melanoma cells (Fig. 1A and Fig. S1). BRAF is an important genetic alteration in melanoma cells. The melanoma cell lines used in this study contain both wild type (C8161.9 and MeWo) and mutant (WM278 and SK-Mel-28) BRAF and no correlation was observed between BRAF-status and RKIP or MDA-9 levels. In addition, we have analyzed two commercially available tissue microarray slides containing sections from metastatic melanoma (n=42) by immunohistochemistry using antibody for MDA-9 and RKIP (Fig. 1B and Fig. S2). Among 35 cases (83%), MDA-9 was strongly expressed whereas RKIP was either completely absent (n=27) or very weekly positive (n=8) providing further confirmative evidence of an inverse correlation between these two proteins. Consistent with these observations, overexpression of MDA-9 by means of a tropism-modified serotype 5/3 adenovirus expressing MDA-9 (Ad.5/3-mda-9) in FM-516 and WM-35 cells resulted in significant downregulation of RKIP expression 48 h post-infection, at both mRNA and protein levels (Fig. 1C–D). Consequently, an RKIP-promoter luciferase reporter had significantly lower activity in metastatic human melanoma cell lines than in FM-516 and WM-35 cells, and in the latter two cells infection with Ad.5/3-mda-9 significantly downregulated RKIP-promoter activity (Fig. 1E). These findings indicate that MDA-9 negatively regulates RKIP expression transcriptionally.

In light of a previous study in prostate cancer (39), we hypothesized and subsequently confirmed through LOF and GOF experiments that Snail, a zinc-transcriptional repressor, plays a critical role in MDA-9-mediated RKIP suppression (Fig. 2A). Consistent with a previous study (40), the MAPK pathway also regulates Snail expression in melanoma cells including both cells with wild-type or mutant BRAF (Fig. 2B and Fig. S3). Based on this consideration, we next investigated how MDA-9 affects Snail expression levels. Consistent with previous observations (41), we found that following contact with fibronectin, ERK was activated by MDA-9 and this activation positively regulated Snail expression (Fig. 2C) subsequently repressing RKIP transcriptionally. For MDA-9-mediated signaling in the context of Src activation, adhesion to fibronectin is important as documented by Boukerche et al., (13). Consistent with our previous observations, MDA-9 activates ERK1/2 on fibronectin-coated plates, but not on plastic plates, which establishes the rational to perform experiments on fibronectin-coated plates (Fig. S4A). However, fibronectin-dependence was not evident in BRAF mutant WM278 and SK-Mel-28 cells (Fig. S4B). To obtain insights relative to persistent overexpression of MDA-9 on RKIP expression in FM-516 cells, several stable mda-9 overexpressing FM-516 clones were established. Two clones, FM-516 mda-9 Cl.10 and Cl.14 express significantly higher levels of mda-9 mRNA and protein compared with parental cells (Fig. S5A and S5B). Consistent with previous findings (8), these clones demonstrated significant growth in soft agar as well as invasiveness as compared with parental cells (Fig. S5C and S5D). As predicted, constitutive RKIP protein and mRNA levels (Fig. S5A and S5B), as well as RKIP-promoter luciferase activity (Fig. S5E), were significantly lower in these two clones compared with parental FM-516 cells.

Restoration of RKIP activity reduces invasiveness and spontaneous melanoma metastasis

To explore the role of RKIP in melanoma invasion and anchorage-independent growth, highly metastatic C8161.9 and FM-516 mda-9 Cl.14 cells were transiently transfected with a HA-tagged RKIP expression plasmid (HA-RKIP). We confirmed that the HA-RKIP was efficiently expressed in different melanoma cells as documented by immunoblotting at 48 h after construct transfection (Fig. S6A). It is relevant to mention that this ectopic expression did not effect cell survival after 48 h (Fig. S6B). As shown in Fig. 3A and 3B, forced expression of RKIP in these two cell types significantly inhibited their anchorage independent growth and invasive properties. In addition, as expected RKIP overexpression resulted in significant changes in cell morphology and a decrease in intracellular actin staining, suggesting a complete disorganization of the actin cytoskeleton (Fig. S7). It is worth noting that mutant BRAF expressing WM278 and SK-Mel-28 cells also responded in a similar manner to RKIP as wild type BRAF C8161.9 expressing melanoma cells (Fig. S8A) documenting an apparent lack of dependence on BRAF status in mediating RKIP effects in melanoma cells.

To examine the effects of RKIP expression in the context of in vivo metastasis, we transiently transfected HA-RKIP into both FM-516 mda-9 Cl.14 and C8161.9 cells and implanted these cells into the chicken chorioallantoic membrane (CAM). To differentiate between the implanted human melanoma cells and chicken embryonic cells we used Alu repeat RT-PCR (Alu PCR). The quantitative nature of the Alu PCR along with its enhanced sensitivity (as low as 10–50 human cells detectable in any given host tissue) provided a means of analyzing the metastatic properties of the cell variants. The primary tumor implanted in the CAM, the invading cells in the lower CAM and metastatic cells in the lung were subjected to Alu PCR. When inoculated at 1 × 10⁵ cells per embryo either cell lines transfected with an empty vector or HA-RKIP did not show any differences in the volume of the primary tumor. However, invasion of the tumor cells into the lower CAM was ~6-fold lower in HA-RKIP transfected cells compared to empty vector-transfected cells (Fig. 3C). Similarly, a significant decrease in lung metastasis was observed upon overexpression of RKIP in both cell types (Fig. 3C).

RKIP suppresses MDA-9-mediated signaling

We previously documented in melanoma cells that upon adhesion to fibronectin MDA-9 activates c-Src through phosphorylation at Tyr⁴¹⁸ leading to FAK phosphorylation and initiates a signaling cascade that culminates in enhanced cell invasion (13, 14). Consistent with previous findings, when seeded on fibronectin-coated plates, all metastatic melanoma cells that constitutively have higher levels of MDA-9 showed by Western blotting a significant up-regulation of Src phosphorylation at Tyr⁴¹⁸ and FAK phosphorylation at Tyr³⁹⁷ as compared with FM-516 or WM35 cells (Fig. 4A). Similar effects were also evident in FM516 mda-9 Cl.14 cells upon adhesion to fibronectin (Fig. 4A). Next, we co-transfected HA-RKIP and mda-9 into FM-516 cells and after 48 h cells were transferred to fibronectin-coated plates and phosphorylation of both Src and FAK was determined. As shown in Fig. 4B, mda-9 alone increased Src and FAK phosphorylation, while HA-RKIP alone did not alter basal levels of Src and FAK phosphorylation, whereas it significantly decreased the mda-9-mediated increases in phosphorylated Src and FAK in FM-516 cells. These experiments also indicate that although MDA-9 suppressed RKIP expression (Fig. 1C and 1D), RKIP expression did not affect MDA-9 expression (Fig. 4B). Silencing of MDA-9 by expressing shRNA of mda-9 in C8161.9 cells significantly reduced Src and FAK activation. Concomitantly, ectopic expression of RKIP did not further alter the level of phospho-Src/FAK indicating that RKIP specifically downregulates MDA-9-triggered Src and FAK phosphorylation. Our observations also reveal that RKIP similarly affects the phospo-Src/FAK levels in BRAF mutant cells (Fig. S8B).

We next studied the effect of RKIP on NF-κB activation, as a consequence of Src and FAK activation, by luciferase reporter assays. As shown in Fig. 4C, transfection of HA-RKIP significantly blocked NF-κB luciferase activity in both FM-516 mda-9 Cl.14 and C8161.9 cells. In FM-516 cells, MDA-9-induced NF-κB luciferase activity, consistent with RKIP function, was down regulated upon co-transfection with HA-RKIP. However, in the presence of mda-9 shRNA, RKIP failed to down regulate NF-κB luciferase activity in C8161.9 cells further supporting the view that the presence of MDA-9 is required to mediate RKIP action.

RKIP physically interacts with MDA-9

Our finding that the presence of MDA-9 is required to mediate the inhibitory action of RKIP supports the hypothesis that these two proteins might physically interact. To obtain insights into this possible interaction, HA-RKIP and different deletion mutants N60, N93, C134 and C93 (Fig. 5A, Upper panel) were transfected into FM-516 mda-9 Cl.14 and C8161.9 cells. Co-Immunoprecipitation studies revealed that RKIP and MDA-9 physically interact (Fig. 5A) and interestingly, this interaction significantly blocks MDA-9’s ability to interact with c-Src (Fig. 5A) supporting the hypothesis that restoration of RKIP might result in competition with c-Src for binding to MDA-9. Specifically, MDA-9 interacts with C134 and N60, both of which contain the RAF-1 binding site (amino acids 77–108) (5, 42) (Fig. 5A). Additionally, expression of these constructs also reduces the endogenous MDA-9 and c-Src interaction as verified by immunoprecipitation and immunoblotting assays. As predicted, the N93 and C93 HA-RKIP mutants, that lack the RAF-1 binding domain, display a significantly reduced interaction with MDA-9 without interrupting the MDA-9 and c-Src interaction (Fig. 5A). Consistent with the immunoprecipitation data, double immunofluorescence using anti-HA and anti-MDA-9 antibodies in HA-RKIP and its different deletion mutants transfected into C8161.9 cells plated on fibronectin also documented co-localization of RKIP and MDA-9, providing further confirmation of interaction between these two proteins (Fig. 5B). As a corollary, C134 and N60 constructs, that interact with MDA-9, downregulated c-Src and FAK phosphorylation in C8161.9 cells while C93 and N93 constructs were inactive (Fig. 5C). Similarly, both C134 and N60 deletion mutants reduced anchorage-independent growth and Matrigel invasion of C8161.9 cells, while C93 and N93 mutants did not affect these phenotypes (Fig. 5C).

PDZ domains bind either the carboxyl-terminal sequences of proteins or internal peptide sequences of their binding partners (35, 43, 44). Since MDA-9 did not require the C-terminus of RKIP for binding, we hypothesized an involvement of internal PDZ ligands in mediating this specific interaction. To explore any preferential area of binding in the RAF-1 site of RKIP, we generated various expression constructs with different point mutations in two class I (HFLV and GTVL) internal ligands within the RAF-1 binding site (amino acids 77–108) and performed binding affinity assays with MDA-9, RAF-1 and cSrc. Interestingly, a disruptive point mutation at amino acid 88 (valine was replaced with lysine, RKIP^V88K) interrupted the interaction with MDA-9 without impairing the binding affinity for RAF-1 (Fig. 6A). As expected, the expression of this construct did not interfere with the endogenous interaction between MDA-9 and c-Src, as shown by both imunoprecipitation and confocal microscopy (Fig. 6A and 6B). In addition, this particular construct has reduced ability to inhibit the anchorage-independent growth and Matrigel invasion of C8161.9 or mda-9 overexpressing FM-516 clones compared to wild type or other mutant RKIP molecules (Fig. 6C) emphasizing the importance of MDA-9 and RKIP interactions in RKIP-mediated suppression of melanoma invasion.

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