The genomic regulation of metastatic dormancy.

The genomics and pathways governing metastatic dormancy are critically important drivers of long-term patient survival given the considerable portion of cancers that recur aggressively months to years after initial treatments. Our understanding of dormancy has expanded greatly in the last two decades, with studies elucidating that the dormant state is regulated by multiple genes, microenvironmental (ME) interactions, and immune components. These forces are exerted through mechanisms that are intrinsic to the tumor cell, manifested through cross-talk between tumor and ME cells including those from the immune system, and regulated by angiogenic processes in the nascent micrometastatic niche. The development of new in vivo and 3D ME models, as well as enhancements to decades-old tumor cell pedigree models that span the development of metastatic dormancy to aggressive growth, has helped fuel what arguably is one of the least understood areas of cancer biology that nonetheless contributes immensely to patient mortality. The current review focuses on the genes and molecular pathways that regulate dormancy via tumor-intrinsic and ME cells, and how groups have envisioned harnessing these therapeutically to benefit patient survival.

Metastasis suppressor genes in clinical practice: are they druggable?

Since the identification of NM23 (now called NME1) as the first metastasis suppressor gene (MSG), a small number of other gene products and non-coding RNAs have been identified that suppress specific parameters of the metastatic cascade, yet which have little or no ability to regulate primary tumor initiation or maintenance. MSG can regulate various pathways or cell biological functions such as those controlling mitogen-activated protein kinase pathway mediators, cell-cell and cell-extracellular matrix protein adhesion, cytoskeletal architecture, G-protein-coupled receptors, apoptosis, and transcriptional complexes. One defining facet of this gene class is that their expression is typically downregulated, not mutated, in metastasis, such that any effective therapeutic intervention would involve their re-expression. This review will address the therapeutic targeting of MSG, once thought to be a daunting task only facilitated by ectopically re-expressing MSG in metastatic cells in vivo. Examples will be cited of attempts to identify actionable oncogenic pathways that might suppress the formation or progression of metastases through the re-expression of specific metastasis suppressors.

AKAP12 Supports Blood-Brain Barrier Integrity against Ischemic Stroke.

A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that associates with intracellular molecules to regulate multiple signal transductions. Although the roles of AKAP12 in the central nervous system are still relatively understudied, it was previously shown that AKAP12 regulates blood-retinal barrier formation. In this study, we asked whether AKAP12 also supports the function and integrity of the blood-brain barrier (BBB). In a mouse model of focal ischemia, the expression level of AKAP12 in cerebral endothelial cells was upregulated during the acute phase of stroke. Also, in cultured cerebral endothelial cells, oxygen-glucose deprivation induced the upregulation of AKAP12. When AKAP12 expression was suppressed by an siRNA approach in cultured endothelial cells, endothelial permeability was increased along with the dysregulation of ZO-1/Claudin 5 expression. In addition, the loss of AKAP12 expression caused an upregulation/activation of the Rho kinase pathway, and treatment of Rho kinase inhibitor Y-27632 mitigated the increase of endothelial permeability in AKAP12-deficient endothelial cell cultures. These in vitro findings were confirmed by our in vivo experiments using Akap12 knockout mice. Compared to wild-type mice, Akap12 knockout mice showed a larger extent of BBB damage after stroke. However, the inhibition of rho kinase by Y-27632 tightened the BBB in Akap12 knockout mice. These data may suggest that endogenous AKAP12 works to alleviate the damage and dysfunction of the BBB caused by ischemic stress. Therefore, the AKAP12-rho-kinase signaling pathway represents a novel therapeutic target for stroke.

A-Kinase Anchor Protein 12 Is Required for Oligodendrocyte Differentiation in Adult White Matter.

Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A-kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild-type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2-months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y-maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12-expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in-vitro OPC maturation. However, conditioned media from AKAP12-deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC-to-oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. Stem Cells 2018;36:751-760.

Origin of parietal podocytes in atubular glomeruli mapped by lineage tracing.

Parietal podocytes are fully differentiated podocytes lining Bowman's capsule where normally only parietal epithelial cells (PECs) are found. Parietal podocytes form throughout life and are regularly observed in human biopsies, particularly in atubular glomeruli of diseased kidneys; however, the origin of parietal podocytes is unresolved. To assess the capacity of PECs to transdifferentiate into parietal podocytes, we developed and characterized a novel method for creating atubular glomeruli by electrocoagulation of the renal cortex in mice. Electrocoagulation produced multiple atubular glomeruli containing PECs as well as parietal podocytes that projected from the vascular pole and lined Bowman's capsule. Notably, induction of cell death was evident in some PECs. In contrast, Bowman's capsules of control animals and normal glomeruli of electrocoagulated kidneys rarely contained podocytes. PECs and podocytes were traced by inducible and irreversible genetic tagging using triple transgenic mice (PEC- or Pod-rtTA/LC1/R26R). Examination of serial cryosections indicated that visceral podocytes migrated onto Bowman's capsule via the vascular stalk; direct transdifferentiation from PECs to podocytes was not observed. Similar results were obtained in a unilateral ureter obstruction model and in human diseased kidney biopsies, in which overlap of PEC- or podocyte-specific antibody staining indicative of gradual differentiation did not occur. These results suggest that induction of atubular glomeruli leads to ablation of PECs and subsequent migration of visceral podocytes onto Bowman's capsule, rather than transdifferentiation from PECs to parietal podocytes.

Proximal tubular cells contain a phenotypically distinct, scattered cell population involved in tubular regeneration.

Regeneration of injured tubular cells occurs after acute tubular necrosis primarily from intrinsic renal cells. This may occur from a pre-existing intratubular stem/progenitor cell population or from any surviving proximal tubular cell. In this study, we characterize a CD24-, CD133-, and vimentin-positive subpopulation of cells scattered throughout the proximal tubule in normal human kidney. Compared to adjacent 'normal' proximal tubular cells, these CD24-positive cells contained less cytoplasm, fewer mitochondria, and no brush border. In addition, 49 marker proteins are described that are expressed within the proximal tubules in a similar scattered pattern. For eight of these markers, we confirmed co-localization with CD24. In human biopsies of patients with acute tubular necrosis (ATN), the number of CD24-positive tubular cells was increased. In both normal human kidneys and the ATN biopsies, around 85% of proliferating cells were CD24-positive - indicating that this cell population participates in tubular regeneration. In healthy rat kidneys, the novel cell subpopulation was absent. However, upon unilateral ureteral obstruction (UUO), the novel cell population was detected in significant amounts in the injured kidney. In summary, in human renal biopsies, the CD24-positive cells represent tubular cells with a deviant phenotype, characterized by a distinct morphology and marker expression. After acute tubular injury, these cells become more numerous. In healthy rat kidneys, these cells are not detectable, whereas after UUO, they appeared de novo - arguing against the notion that these cells represent a pre-existing progenitor cell population. Our data indicate rather that these cells represent transiently dedifferentiated tubular cells involved in regeneration.

PTEN-regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression.

PTEN loss, one of the most frequent mutations in prostate cancer (PC), is presumed to drive disease progression through AKT activation. However, two transgenic PC models with Akt activation plus Rb loss exhibited different metastatic development: Pten/RbPE:-/- mice produced systemic metastatic adenocarcinomas with high AKT2 activation, whereas RbPE:-/- mice deficient for the Src-scaffolding protein, Akap12, induced high-grade prostatic intraepithelial neoplasias and indolent lymph node dissemination, correlating with upregulated phosphotyrosyl PI3K-p85α. Using PC cells isogenic for PTEN, we show that PTEN-deficiency correlated with dependence on both p110ß and AKT2 for in vitro and in vivo parameters of metastatic growth or motility, and with downregulation of SMAD4, a known PC metastasis suppressor. In contrast, PTEN expression, which dampened these oncogenic behaviors, correlated with greater dependence on p110α plus AKT1. Our data suggest that metastatic PC aggressiveness is controlled by specific PI3K/AKT isoform combinations influenced by divergent Src activation or PTEN-loss pathways.

Gravin orchestrates protein kinase A and ß2-adrenergic receptor signaling critical for synaptic plasticity and memory.

A kinase-anchoring proteins (AKAPs) organize compartmentalized pools of protein kinase A (PKA) to enable localized signaling events within neurons. However, it is unclear which of the many expressed AKAPs in neurons target PKA to signaling complexes important for long-lasting forms of synaptic plasticity and memory storage. In the forebrain, the anchoring protein gravin recruits a signaling complex containing PKA, PKC, calmodulin, and PDE4D (phosphodiesterase 4D) to the ß2-adrenergic receptor. Here, we show that mice lacking the α-isoform of gravin have deficits in PKA-dependent long-lasting forms of hippocampal synaptic plasticity including ß2-adrenergic receptor-mediated plasticity, and selective impairments of long-term memory storage. Furthermore, both hippocampal ß2-adrenergic receptor phosphorylation by PKA, and learning-induced activation of ERK in the CA1 region of the hippocampus are attenuated in mice lacking gravin-α. We conclude that gravin compartmentalizes a significant pool of PKA that regulates learning-induced ß2-adrenergic receptor signaling and ERK activation in the hippocampus in vivo, thereby organizing molecular interactions between glutamatergic and noradrenergic signaling pathways for long-lasting synaptic plasticity, and memory storage.

Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated.

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.

Suppression of tumor and metastasis progression through the scaffolding functions of SSeCKS/Gravin/AKAP12.

Scaffolding proteins such as SSeCKS/Gravin/AKAP12 ("AKAP12") are thought to control oncogenic signaling pathways by regulating key mediators in a spatiotemporal manner. The downregulation of AKAP12 in many human cancers, often associated with promoter hypermethylation, or the loss of its locus at 6q24-25.2, correlates with progression to malignancy and metastasis. The forced re-expression of AKAP12 in cancer cell lines suppresses in vitro parameters of oncogenic growth, invasiveness, and cell motility through its ability to scaffold protein kinase C (PKC), F-actin, cyclins, Src, and phosphoinositides, and possibly through additional scaffolding domains for PKA, calmodulin, ß1,4-galactosyltransferase-polypeptide-1, ß2-adrenergic receptors, and cAMP-specific 3',5'-cyclic phosphodiesterase 4D. Moreover, AKAP12 re-expression in tumor models results in metastasis suppression through the inhibition of Src-regulated, VEGF-mediated neovascularization at distal sites. The current review will describe the emerging understanding of how AKAP12 regulates cellular senescence and oncogenic progression at the level of tumor cells and tumor-associated microenvironment via its multiple scaffolding functions.

Role of A-Kinase Anchoring Protein 12 in the Central Nervous System.

A-kinase anchoring protein (AKAP) 12 is a scaffolding protein that anchors various signaling proteins to the plasma membrane. These signaling proteins include protein kinase A, protein kinase C, protein phosphatase 2B, Src-family kinases, cyclins, and calmodulin, which regulate their respective signaling pathways. AKAP12 expression is observed in the neurons, astrocytes, endothelial cells, pericytes, and oligodendrocytes of the central nervous system (CNS). Its physiological roles include promoting the development of the blood-brain barrier, maintaining white-matter homeostasis, and even regulating complex cognitive functions such as long-term memory formation. Under pathological conditions, dysregulation of AKAP12 expression levels may be involved in the pathology of neurological diseases such as ischemic brain injury and Alzheimer's disease. This minireview aimed to summarize the current literature on the role of AKAP12 in the CNS.

Phosphohistidine signaling promotes FAK-RB1 interaction and growth factor-independent proliferation of esophageal squamous cell carcinoma.

Current clinical therapies targeting receptor tyrosine kinases including focal adhesion kinase (FAK) have had limited or no effect on esophageal squamous cell carcinoma (ESCC). Unlike esophageal adenocarcinomas, ESCC acquire glucose in excess of their anabolic need. We recently reported that glucose-induced growth factor-independent proliferation requires the phosphorylation of FAKHis58. Here, we confirm His58 phosphorylation in FAK immunoprecipitates of glucose-stimulated, serum-starved ESCC cells using antibodies specific for 3-phosphohistidine and mass spectrometry. We also confirm a role for the histidine kinase, NME1, in glucose-induced FAKpoHis58 and ESCC cell proliferation, correlating with increased levels of NME1 in ESCC tumors versus normal esophageal tissues. Unbiased screening identified glucose-induced retinoblastoma transcriptional corepressor 1 (RB1) binding to FAK, mediated through a "LxCxE" RB1-binding motif in FAK's FERM domain. Importantly, in the absence of growth factors, glucose increased FAK scaffolding of RB1 in the cytoplasm, correlating with increased ESCC G1→S phase transition. Our data strongly suggest that this glucose-mediated mitogenic pathway is novel and represents a unique targetable opportunity in ESCC.

PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression.

PTEN loss, one of the most frequent mutations in prostate cancer (PC), is presumed to drive disease progression through AKT activation. However, two transgenic PC models with Akt activation plus Rb loss exhibited different metastasis development: Pten/RbPE:-/- mice produced systemic metastatic adenocarcinomas with high AKT2 activation, whereas RbPE:-/- mice deficient for the Src-scaffolding protein, Akap12, induced high-grade prostatic intraepithelial neoplasias and indolent lymph node disseminations, correlating with upregulated phosphotyrosyl PI3K-p85α. Using PC cells isogenic for PTEN, we show that PTEN-deficiency correlated with dependence on both p110ß and AKT2 for in vitro and in vivo parameters of metastatic growth or motility, and with downregulation of SMAD4, a known PC metastasis suppressor. In contrast, PTEN expression, which dampened these oncogenic behaviors, correlated with greater dependence on p110α plus AKT1. Our data suggest that metastatic PC aggressiveness is controlled by specific PI3K/AKT isoform combinations influenced by divergent Src activation or PTEN-loss pathways.

A small molecule focal adhesion kinase (FAK) inhibitor, targeting Y397 site: 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide effectively inhibits FAK autophosphorylation activity and decreases cancer cell viability, clonogenicity and tumor growth in vivo.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in most solid types of tumors and plays an important role in the survival signaling. Recently, we have developed a novel computer modeling combined with a functional assay approach to target the main autophosphorylation site of FAK (Y397). Using these approaches, we identified 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide, called Y11, a small molecule inhibitor targeting Y397 site of FAK. Y11 significantly and specifically decreased FAK autophosphorylation, directly bound to the N-terminal domain of FAK. In addition, Y11 decreased Y397-FAK autophosphorylation, inhibited viability and clonogenicity of colon SW620 and breast BT474 cancer cells and increased detachment and apoptosis in vitro. Moreover, Y11 significantly decreased tumor growth in the colon cancer cell mouse xenograft model. Finally, tumors from the Y11-treated mice demonstrated decreased Y397-FAK autophosphorylation and activation of poly (ADP ribose) polymerase and caspase-3. Thus, targeting the major autophosphorylation site of FAK with Y11 inhibitor is critical for development of cancer therapeutics and carcinogenesis field.

Control of protein kinase C activity, phorbol ester-induced cytoskeletal remodeling, and cell survival signals by the scaffolding protein SSeCKS/GRAVIN/AKAP12.

The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely through its ability to scaffold multiple signaling mediators such as PKC, PKA, cyclins, calmodulin, and Src. Although SSeCKS and PKCα bind phosphatidylserine, we demonstrate that phosphatidylserine-independent binding of PKC by SSeCKS is facilitated by two homologous SSeCKS motifs, EG(I/V)(T/S)XWXSFK(K/R)(M/L)VTP(K/R)K(K/R)X(K/R)XXXEXXXE(E/D) (amino acids 592-620 and 741-769). SSeCKS binding to PKCα decreased kinase activity and was dependent on the two PKC-binding motifs. SSeCKS scaffolding of PKC was increased in confluent cell cultures, correlating with significantly increased SSeCKS protein levels and decreased PKCα activity, suggesting a role for SSeCKS in suppressing PKC activation during contact inhibition. SSeCKS-null mouse embryo fibroblasts displayed increased relative basal and phorbol ester (phorbol 12-myristate 13-acetate)-induced PKC activity but were defective in phorbol 12-myristate 13-acetate-induced actin cytoskeletal reorganization and cell shape change; these responses could be rescued by the forced expression of full-length SSeCKS but not by an SSeCKS variant deleted of its PKC-binding domains. Finally, the PKC binding sites in SSeCKS were required to restore cell rounding and/or decreased apoptosis in phorbol ester-treated LNCaP, LNCaP-C4-2, and MAT-LyLu prostate cancer cells. Thus, PKC-mediated remodeling of the actin cytoskeleton is likely regulated by the ability of SSeCKS to control PKC signaling and activity through a direct scaffolding function.

SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus.

Glomerular parietal epithelial cells (PECs) are precursors to podocytes in mature glomeruli; however, as progenitors, the distinct intrinsic mechanisms that allow for repeated periods of cell-cycle arrest and re-entry of PECs after glomerulogenesis are unknown. Here, we show that the Src-suppressed protein kinase C substrate (SSeCKS), a multivalent scaffolding A kinase anchoring protein, sequesters cyclin D1 in the cytoplasm of quiescent PECs. SSeCKS expression is induced in embryonic PECs, but not in embryonic podocytes, starting at the S phase of glomerulogenesis, and is constitutively expressed postnatally by PECs, but not podocytes, in normal glomeruli. Cyclin D1 was immunoprecipitated with SSeCKS from capsulated glomeruli containing PECs, whereas decapsulated glomeruli without PECs lacked SSeCKS and cyclin D1. Cell-cell contact inhibition of proliferation in cultured PECs induced SSeCKS expression and binding of cyclin D1 by SSeCKS in the cytoplasm, whereas phosphorylation of SSeCKS by activated protein kinase C disrupted binding, resulting in nuclear translocation of cyclin D1. SSeCKS(-/-) mice showed hyperplasia of PECs in otherwise normal glomeruli and developed significantly worse proteinuric glomerular disease, marked by increased PEC proliferation and expression of nuclear cyclin D1, from nephrotoxic nephritis. These results suggest that SSeCKS controls the localization and activity of cyclin D1 in PECs and influences proliferative injury in the glomerulus.

Targeting PIM2 by JP11646 results in significant antitumor effects in solid tumors.

Proviral integration of Moloney virus 2 (PIM2) is a pro­survival factor of cancer cells and a possible therapeutic target in hematological malignancies. However, the attempts at inhibiting PIM2 have yielded underwhelming results in early clinical trials on hematological malignancies. Recently, a novel pan­PIM inhibitor, JP11646, was developed. The present study examined the utility of targeting PIM2 in multiple solid cancers and investigated the antitumor efficacy and the mechanisms of action of JP11646. When PIM2 expression was compared between normal and cancer tissues in publicly available datasets, PIM2 was found to be overexpressed in several types of solid cancers. PIM2 ectopic overexpression promoted tumor growth in in vivo xenograft breast cancer mouse models. The pan­PIM inhibitor, JP11646, suppressed in vitro cancer cell proliferation in a concentration­dependent manner in multiple types of cancers; a similar result was observed with siRNA­mediated PIM2 knockdown, as well as an increased in cell apoptosis. By contrast, another pan­PIM inhibitor, AZD1208, suppressed the expression of downstream PIM2 targets, but not PIM2 protein expression, corresponding to no apoptosis induction. As a mechanism of PIM2 protein degradation, it was found that the proteasome inhibitor, bortezomib, reversed the apoptosis induced by JP11646, suggesting that PIM2 degradation by JP11646 is proteasome­dependent. JP11646 exhibited significant anticancer efficacy with minimal toxicities at the examined doses and schedules in multiple in vivo mice xenograft solid cancer models. On the whole, the present study demonstrates that PIM2 promotes cancer progression in solid tumors. JP11646 induces apoptosis at least partly by PIM2 protein degradation and suppresses cancer cell proliferation in vitro and in vivo. JP11646 may thus be a possible treatment strategy for multiple types of solid cancers.

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

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.

Role for transcription factor TFII-I in the suppression of SSeCKS/Gravin/Akap12 transcription by Src.

The SSeCKS/Gravin/AKAP12 gene, encoding a kinase scaffolding protein with metastasis-suppressing activity, is transcriptionally downregulated in Src-transformed cells through the recruitment of HDAC1 to a Src-responsive proximal promoter site charged with Sp1, Sp3 and USF1. However, the ectopic expression of these proteins formed a suppressive complex in Src-transformed but not in parental NIH3T3 cells, suggesting the involvement of additional repressor factors. Transcription factor II-I (TFII-I) [general transcription factor 2i (Gtf2i)] was identified by mass spectrometry as being associated with the SSeCKS promoter complex in NIH3T3/Src cells, and moreover, the Src-induced tyrosine phosphorylation of TFII-I significantly increased its binding to the SSeCKS proximal promoter. siRNA-mediated knockdown of TFII-I or the expression of TFII-I(Y248/249F) caused the derepression of SSeCKS in NIH3T3/Src cells. Taken with previous data showing that the tyrosine phosphorylation of TFII-I facilitates its nuclear translocation, these data suggest that Src-family kinase-mediated phosphorylation converts a portion of TFII-I into a transcriptional repressor.

Carcinogen-induced squamous papillomas and oncogenic progression in the absence of the SSeCKS/AKAP12 metastasis suppressor correlate with FAK upregulation.

The ability of SSeCKS/Gravin/AKAP12 (SSeCKS) to negatively regulate cell cycle progression is thought to relate to its spatiotemporal scaffolding activity for key signaling molecules such as protein kinase A and C, calmodulin and cyclins. SSeCKS is downregulated upon progression to malignancy in many cancer types, including melanoma and nonmelanoma skin cancer. The forced re-expression of SSeCKS is especially potent in suppressing metastasis through the inhibition of VEGF-mediated neovascularization. We have previously shown that SSeCKS-null (KO) mice exhibit hyperplasia and focal dysplasia in the prostate marked by activated Akt. To address whether KO mice exhibit increased skin carcinogenesis, WT and KO C57BL/6 mice were treated topically with 12-O-tetradecanoylphorbol-13-acetate and 7,12-dimethylbenzanthracene. Compared to WT mice, KO mice developed squamous papillomas more rapidly and in greater numbers and also exhibited significantly increased progression to squamous cell carcinoma. Untreated KO epidermal layers were thicker than those in age-matched WT mice and exhibited significantly increased levels of FAK and phospho-ERK1/2, known mediators of carcinogen-induced squamous papilloma progression to carcinoma. Compared to protein levels in WT mouse embryo fibroblasts (MEF), SSeCKS levels were increased in FAK-null cells, whereas FAK levels were increased in SSeCKS-null cells. RNAi studies in WT MEF cells suggest that SSeCKS and FAK attenuate each other's expression. Our study implicates a role for SSeCKS in preventing of skin cancer progression possibly through negatively regulating FAK expression.