SSeCKS/Gravin/AKAP12 inhibits cancer cell invasiveness and chemotaxis by suppressing a protein kinase C- Raf/MEK/ERK pathway

Abstract

SSeCKS/Gravin/AKAP12 ("SSeCKS") encodes a cytoskeletal protein that regulates G(1) --> S progression by scaffolding cyclins, protein kinase C (PKC) and PKA. SSeCKS is down-regulated in many tumor types including prostate, and when re-expressed in MAT-LyLu (MLL) prostate cancer cells, SSeCKS selectively inhibits metastasis by suppressing neovascularization at distal sites, correlating with its ability to down-regulate proangiogenic genes including Vegfa. However, the forced re-expression of VEGF only rescues partial lung metastasis formation. Here, we show that SSeCKS potently inhibits chemotaxis and Matrigel invasion, motility parameters contributing to metastasis formation. SSeCKS suppressed serum-induced activation of the Raf/MEK/ERK pathway, resulting in down-regulation of matrix metalloproteinase-2 expression. In contrast, SSeCKS had no effect on serum-induced phosphorylation of the Src substrate, Shc, in agreement with our previous data that SSeCKS does not inhibit Src kinase activity in cells. Invasiveness and chemotaxis could be restored by the forced expression of constitutively active MEK1, MEK2, ERK1, or PKCalpha. SSeCKS suppressed phorbol ester-induced ERK1/2 activity only if it encoded its PKC binding domain (amino acids 553-900), suggesting that SSeCKS attenuates ERK activation through a direct scaffolding of conventional and/or novel PKC isozymes. Finally, control of MLL invasiveness by SSeCKS is influenced by the actin cytoskeleton: the ability of SSeCKS to inhibit podosome formation is unaffected by cytochalasin D or jasplakinolide, whereas its ability to inhibit MEK1/2 and ERK1/2 activation is nullified by jasplakinolide. Our findings suggest that SSeCKS suppresses metastatic motility by disengaging activated Src and then inhibiting the PKC-Raf/MEK/ERK pathways controlling matrix metalloproteinase-2 expression and podosome formation.

FIGURE 1.

SSeCKS suppresses chemotaxis and Matrigel invasion of metastatic prostate cancer cells. A, chemotaxis. MLL/tet-SSeCKS cells were seeded atop Boyden chamber membranes in the presence or absence of tet (− or + SSeCKS, respectively) with 5% BS in the bottom well as the chemoattractant (top left panels) or no serum, as a negative control (bottom left panels). After 12 h, the cells were fixed, stained, and counted as described under “Experimental Procedures.” Six separate microscopic fields on the stained membranes from duplicate experiments were counted to determine the average number of cells/field (error bar = S.D.). *, p < 0.005. B, invasion. The ability of MLL/tet-SSeCKS cells to invade after 24 h through 1-mm growth factor-reduced Matrigel barriers atop Boyden chamber membranes was assessed. Six separate microscopic fields on the stained membranes from duplicate experiments were counted to determine the average number of cells/field (error bar = S.D.). *, p < 0.01. C, immunoblot (IB) of SSeCKS and actin protein levels from MLL/tet-SSeCKS cells grown in the presence or absence of tet (− or + SSeCKS, respectively).

FIGURE 2.

SSeCKS inhibits MMP-2 expression and secretion. A, conditioned medium obtained from an overnight incubation of equal numbers of MLL/tet-SSeCKS cells grown in serum-free DMEM +/− tet was subjected to zymography as described under “Experimental Procedures.” Controls include overnight medium from NIH3T3/v-Src cells and purified MMP-2 and -9. The 95- and 67-kDa MMP-9 and -2, respectively, are identified by arrows as well as the unactivated form of MMP-2 (pro-MMP-2). Aliquots of purified, enzymatically active MMP-2 and -9 were run as controls. B, RNA derived from MLL/tet-SSeCKS cells grown in serum-free DMEM +/− tet was subjected to semi-quantitative reverse transcriptase-PCR using primer sets for MMP-2, MT1-MMP, TIMP2, and β-actin as described under “Experimental Procedures.” The results are typical of at least three independent experiments.

FIGURE 3.

Inhibition of chemotaxis and invasiveness by SSeCKS correlates with suppression of serum-induced MEK-1/2 and ERK-1/2 activation. A, re-expression of SSeCKS attenuates MEK/ERK. Serum-starved MLL/tet-SSeCKS cells (+/− tet) were stimulated with 10% BS in DMEM for various times, and then RIPA lysates were analyzed by IB (40 μg of protein/lane) for SSeCKS (to show inducibility in the absence of tet), phospho- and apo-forms of MEK1/2 and ERK1/2, and actin (protein loading control). B, graphic representation of the relative MEK1/2 and ERK1/2 activation levels (phospho-protein levels normalized to total MEK1/2 or ERK1/2 protein levels, with basal levels set arbitrarily at 1, as shown by the dotted lines). Similar effects by serum and SSeCKS were identified in two other independent experiments (error bar = S.D.). C, Boyden chamber chemotaxis and invasion assays as described in the legend to Fig. 1 were preformed in the presence of U0126 (10 μm) or vehicle in the presence (black columns) or absence (white columns) of tet (error bar = S.D. from two independent experiments).

FIGURE 4.

Activated MEK rescues SSeCKS-suppressed chemotaxis and invasion. A, CA-MEK1 or -MEK2 were transiently co-transfected along with pEGFP into MLL/tet-SSeCKS cells. The IB analysis shows increased MEK1 or MEK2 protein levels in cells expressing CA-MEK1 or CA-MEK2 (versus vector alone), respectively. The CA-MEK alleles induced increased phosphorylation of ERK1/2 as measured by the relative pro-ERK1/2 levels. GFP protein levels are shown as a loading control. (Note that the cell lysates expressing CA-MEK alleles could not be probed for changes in phospho-MEK levels because the CA-MEK products contain amino acid substitutions at the site recognized by phospho-specific MEK Abs (Ser^217/221).) B, chemotaxis and invasion assays were performed as described in the legend to Fig. 1 comparing MLL/tet-SSeCKS/vector (−) versus MLL/tet-SSeCKS/CA-MEK (+) grown in the presence or absence of tet (error bar = S.D. from two independent experiments). C, CA-MEK1 or CA-MEK2 rescues MMP-2 expression in SSeCKS re-expressing MLL cells. Conditioned medium from serum-starved MLL/tet-SSeCKS/vector (−) versus MLL/tet-SSeCKS/CA-MEK (+) grown in the presence or absence of tet were assessed for MMP-2 activity by zymography as described in the legend to Fig. 2. D, SSeCKS-induced cell flattening is suppressed by the forced expression of CA-MEK1 or CA-MEK2. Round (R) or flat (F) transfected cells (GFP-positive) were identified by fluorescence (panel D, top row) plus phase-contrast microscopy (panel D, bottom row). Size bar = 5 μm. E, the percentage of round cells in at least three independent fields from panel D (>50 cells/field). Error bars = S.D.

FIGURE 5.

SSeCKS inhibits podosome formation and induces stress fiber formation. A, MLL/tet-SSeCKS cells grown in the presence or absence of tet, and in the presence of tet with U0126 cells were imaged by phase-contrast microscopy (left column) or fixed and stained for F-actin (rhodamine-phalloidin) stress fibers (SF) (middle column), and focal adhesions (vinculin, right column). Podosomes (P) are defined as enrichments of F-actin on the cell surface, often exhibiting a “ring” structure. Size bars = 5 μm. B, SSeCKS re-expression decreases total podosome formation. The percentage of cells exhibiting podosomes (left) or the frequency of podosomes per cell (right) was quantified as the average of three separate visual fields containing roughly 30 cells per field; error bars = S.E. *, p < 0.01; **, p < 0.05.

FIGURE 6.

Inhibition of serum-induced c-Raf and PKCδ activation by SSeCKS. Serum-starved MLL/tet-SSeCKS cells (+/− tet) were stimulated with 10% BS in DMEM for various times, and then RIPA lysates were analyzed by IB (30 μg of protein/lane) for phospho- and apo-forms of c-Raf and Shc, PKCδ and GAPDH. CF, cleaved fragment identifying active PKCδ.

FIGURE 7.

Activated PKCα rescues SSeCKS-suppressed invasion and -induced cell flattening. A, re-expression of SSeCKS decreases PKC activity. Lysates from MLL/tet-SSeCKS cells grown overnight in +/− tet conditions in the absence of serum were analyzed for total PKC kinase activity as described under “Experimental Procedures.” Error bars = S.D. of triplicates, with the mean PKC activity in the absence of SSeCKS re-expression set at 1.0. B, IB analysis of 293T cell lysates transiently co-transfected with CA-PKCα(HA) plus pEGFP, probed for PKCα, HA, or ERK1/2^{pro-Thr-202/Tyr-204}. C, SSeCKS-induced cell flattening is suppressed by forced expression of CA-PKCα. The percentage of round cells in at least three independent fields is shown. Error bars = S.D. D, CA-PKCα rescues Matrigel invasion of SSeCKS re-expressing MLL. MLL/tet-SSeCKS cells transiently transfected with CA-PKCα plus pEGFP were grown in + or − tet conditions and then subjected to Matrigel invasion assays as described in the legend to Fig. 1B except that only GFP-positive invasive cells were scored. Error bars = S.D. of triplicate wells.

FIGURE 8.

SSeCKS inhibits PMA-induced ERK1/2 activation via scaffolding of PKC. A, re-expression of SSeCKS decreases PMA-induced ERK1/2 activity. Serum-starved MLL/tet-SSeCKS cells (+/− tet) were stimulated with 100 nm PMA in DMEM for various times, and then RIPA lysates were analyzed by IB (30 μg of protein/lane) for phospho-ERK1/2, ERK1/2, and GAPDH. B, full-length (FL), but not Δ553–900 SSeCKS, suppresses PMA-induced ERK1/2 activity. Lysates of 293T cells transiently transfected with pEGFP control vector or expression vectors encoding GFP fusions of FL or Δ553–900 SSeCKS, treated with 100 nm PMA for 15 min, were probed by IB analysis for GFP, phospho-ERK1/2, ERK1/2, and GAPDH.