SSeCKS/Gravin/AKAP12 attenuates expression of proliferative and angiogenic genes during suppression of v-Src-induced oncogenesis

BACKGROUND: SSeCKS is a major protein kinase C substrate with kinase scaffolding and metastasis-suppressor activity whose expression is severely downregulated in Src- and Ras-transformed fibroblast and epithelial cells and in human prostate, breast, and gastric cancers. We previously used NIH3T3 cells with tetracycline-regulated SSeCKS expression plus a temperature-sensitive v-Src allele to show that SSeCKS re-expression inhibited parameters of v-Src-induced oncogenic growth without attenuating in vivo Src kinase activity. METHODS: We use cDNA microarrays and semi-quantitative RT-PCR analysis to identify changes in gene expression correlating with i) SSeCKS expression in the absence of v-Src activity, ii) activation of v-Src activity alone, and iii) SSeCKS re-expression in the presence of active v-Src. RESULTS: SSeCKS re-expression resulted in the attenuation of critical Src-induced proliferative and pro-angiogenic gene expression including Afp, Hif-1α, Cdc20a and Pdgfr-β, and conversely, SSeCKS induced several cell cycle regulatory genes such as Ptpn11, Gadd45a, Ptplad1, Cdkn2d (p19), and Rbbp7. CONCLUSION: Our data provide further evidence that SSeCKS can suppress Src-induced oncogenesis by modulating gene expression downstream of Src kinase activity

Src controls castration recurrence of CWR22 prostate cancer xenografts

Recurrence of prostate cancer (CaP) after androgen-deprivation therapy continues to have the greatest impact on patient survival. Castration-recurrent (CR)-CaP is likely driven by the activation of androgen receptor (AR) through multiple mechanisms including induction of AR coregulators, AR mutants or splice variants, and AR posttranslational modification such as phosphorylation by Src-family and Ack1 tyrosine kinases. Here, we address whether Src is required for the CR growth of human CWR22 CaP xenografts. The shRNA-mediated Src knockdown or treatment with the Src inhibitors, dasatinib or KXO1, reduced CaP recurrence over controls and increased time-to-recurrence following castration. Moreover, CR-CaP [Src-shRNA] tumors that recurred had similar Src protein and activation levels as those of parental cells, strengthening the notion that Src activity is required for progression to CR-CaP. In contrast, the ability of dasatinib or KXO1 to inhibit Src kinase activity in vitro did not correlate with their ability to inhibit serum-driven in vitro proliferation of CR and androgen-dependent stable cell lines derived from CWR22 tumors (CWR22Rv1 and CWR22PC, respectively), suggesting that the in vitro proliferation of these CaP lines is Src independent. Taken together, these findings strongly suggest that Src is a potent and specific therapeutic target for CR-CaP progression

SSeCKS/Gravin/AKAP12 attenuates expression of proliferative and angiogenic genes during suppression of v-Src-induced oncogenesis-1

<p><b>Copyright information:</b></p><p>Taken from "SSeCKS/Gravin/AKAP12 attenuates expression of proliferative and angiogenic genes during suppression of v-Src-induced oncogenesis"</p><p>BMC Cancer 2006;6():105-105.</p><p>Published online 25 Apr 2006</p><p>PMCID:PMC1463002.</p><p>Copyright © 2006 Liu et al; licensee BioMed Central Ltd.</p> or from HCT116 or HT29 (Panel B) were subjected to RT-PCR analysis as described in Materials and Methods using primer sets described in Table 1. These results are typical of at least two independent experiments

Suppression of Chemotaxis by SSeCKS via Scaffolding of Phosphoinositol Phosphates and the Recruitment of the Cdc42 GEF, Frabin, to the Leading Edge

<div><p>Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (Src^poY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.</p></div

A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness

SSeCKS inhibits chemotaxis and affects leading edge protrusions.

<p><i>A</i>, Relative chemotaxis of MEF (WT or KO), DU145 (transfected with control [con] or human SSeCKS-siRNA [si]) and MDA-MB-231 cells (transfected with empty vector [V] or an SSeCKS-GFP expression plasmid [SS]), as measured in Boyden chamber assays using serum as the chemoattractant. <i>Error bars</i>, S.E. of triplicate assays. *, p<0.02, **, p<0.005. <i>B</i>, IB analysis of SSeCKS and GAPDH levels in the cells described in Panel A. <i>C</i>, Relative chemotaxis of WT or KO MEF using PDGF as the chemoattractant in Boyden chamber assays. <i>Error bars</i>, S.E. of three wounding fields in two independent experiments. **, p<0.02. <i>D,</i> Relative ability of WT or KO MEF to close wound scratches, based on measuring three wound field gaps at a given time in triplicate experiments. <i>E</i>, Agarose chemotaxis spot assay. <i>Top: left panel-</i> cartoon of motile cells (<i>black</i>) moving towards (<i>red arrows</i>) an agarose spot containing chemoattractant; <i>right panels-</i> example of assay without (“culture media” plus PBS; <i>left</i>) or with chemoattractant gradient (“serum-free media” plus EGF/PDGF in spot; <i>right</i>). <i>Bottom:</i> Leading edges of chemotactic WT cells predominantly display lamellipodia whereas those of KO cells predominantly display filopodia-like extensions. <i>Left panels-</i> phase contrast microscopy of chemotactic cells. <i>Open-head black arrows</i>, filopodia; <i>closed-head black arrows</i>, lamellipodia; <i>white arrows</i>, chemotaxis direction. <i>Middle and right panels-</i> IFA staining of SSeCKS or F-actin in WT or KO MEF. <i>Arrows</i>, chemotaxis direction. <i>Scale bar</i>, 10 µm. <i>F</i>, Fraction of chemotactic WT or KO cells with leading edge lamellipodia or filopodia. <i>Error bars</i>, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. *, p<0.02, **, p<0.005. <i>G</i>, Percentage of chemotactic WT or KO cells in Panel F with <1, 1–3 or >3 filopodia/leading edge. **, p<0.005. <i>H,</i> Induction of lamellipodia formation in MDA-MB-231 cells transfected with SSeCKS-GFP (<i>vs. GFP vector alone</i>), as shown by IFA for GFP (<i>left panels</i>) or F-actin (<i>center</i>), or following quantification (<i>graph, right</i>). <i>Arrows</i>, chemotaxis direction. <i>Scale bar</i>, 10 µm. <i>Error bars</i>, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. <i>I</i>, IFA for fascin and F-actin in WT and KO MEF. <i>Short arrows</i>, fascin-staining filopodia. <i>Long arrows,</i> chemotaxis direction.</p

Enrichment of activated Src at the tips of leading edge filopodia in chemotactic KO MEF.

<p>IFA analysis of FAK and F-actin (<i>A</i>), or Src^poY416 and F-actin (<i>B</i>) in chemotactic WT and KO MEF. Total levels of FAK or Src^poY416 in the WT vs. KO MEF did not differ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111534#pone.0111534-Akakura1" target="_blank">[28]</a>. <i>Scale bar</i>, 10 µm. <i>Arrow</i>, chemotaxis direction. <i>C,</i> Relative chemotaxis of WT MEF or KO MEF transfected with vector (V), FL-, ΔPBD- or ΔSrc-SSeCKS-GFP. <i>Error bars</i>, S.E. of 5 visual microscope fields with at least 10 cells/field in two independent experiments. **, p<0.005; n.s., not significant. <i>D,</i> Chemotaxis (migrated cells/field) of WT cells treated with vehicle or SKI-606. <i>Error bars</i>, S.E. of 3 independent experiments. *, p<0.02. <i>E,</i> IFA analysis of Frabin and F-actin in KO MEF treated with SKI-606. <i>Scale bar</i>, 10 µm. Arrow, chemotaxis direction. <i>F,</i> Chemotaxis of KO MEF transfected with vector (–) or CA-PI3K and/or treated with SKI-606. <i>Error bars</i>, S.E. of 3 independent experiments. *, p<0.02; n.s., not significant. <i>G,</i> IFA analysis of GFP or Frabin in KO MEF transfected with pEGFP and CA-PI3K and then treated with vehicle or SKI-606. <i>Scale bar</i>, 10 µm. <i>Arrow</i>, chemotaxis direction. <i>H,</i> KO MEF transiently transfected with pEGFP alone or with plasmids encoding CA-PI3K or ΔSrc-SSeCKS, were treated with either vehicle or SKI-606 (1 µM) for 18 h, then subjected to directional chemotaxis assays, fixed and stained for F-actin. <i>Arrows</i>, chemotactic direction.</p

SSeCKS inhibits the rate and directionality of chemotactic cells.

<p><i>A</i>, Hypothetical chemotactic paths of three cells (square, diamond or triangle) based on five time measurements (numbered). The forward migration index (FMI) is calculated as the distance “h”, if a cell theoretically travelled directly towards the chemoattractant source over time-points 1 to 5, divided by “b”, the direct vector from the cell’s start (time-point 1) to end (time-point 5). A cell moving in a straight, direct line towards a chemoattractant would have an FMI = 1. KO MEF have increased FMI (<i>B</i>) and velocity (<i>C</i>) compared to WT MEF. *, p<0.05, **, p<0.01 for at least 20 cells/time-point/condition.</p

Enrichment of Frabin at the leading edge of KO MEF directs increased chemotaxis.

<p><i>A</i>, IFA for Frabin or F-actin in resting vs. chemotactic (<i>arrows</i>) WT and KO MEF. <i>Scale bar,</i> 10 µm. <i>B</i>, IFA of GFP or Frabin in KO MEF re-expressing FL-, ΔPBD-, or ΔSrc-SSeCKS-GFP. <i>Scale bar,</i> 10 µm. <i>Arrow</i>, chemotaxis direction. <i>C</i>, IB analysis of SSeCKS, Frabin or Gapd levels in WT vs. KO MEF, relative to markers (<i>left</i>). <i>D,</i> IB analysis of Frabin or Gapdh protein levels in KO MEF cell lysates transfected with scrambled (scr) or Frabin-specific siRNA. <i>E</i>, IFA analysis of F-actin in KO MEF transfected with scr- or Frabin-siRNA. <i>Scale bar,</i> 5 µm. <i>Arrow</i>, chemotaxis direction. <i>F</i>, Quantification of KO MEF with leading edge lamellipodia vs. filopodia after transfection with scr- or Frabin-siRNA. <i>Error bars</i>, S.E. of 5 visual microscope fields with at least 10 cells/field in two independent experiments. **, p<0.005. <i>G</i>, Effect of scr- or Frabin-siRNA on WT or KO MEF chemotaxis. <i>Error bars</i>, S.E. of triplicates from two independent experiments. **, p<0.01.</p