Shin Akakura

Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein that encodes metastasis-suppressor activity through the suppression of Src-mediated oncogenic signaling and vascular endothelial growth factor expression. SSeCKS expression is down-regulated in Src- and Ras-transformed fibroblasts, in human cancer cell lines and in several types of human cancer, including prostate. Normal human and mouse prostates express abundant SSeCKS in secretory epithelial cells and, to a lesser extent, in the surrounding mesenchyme. Here, we show that the loss of SSeCKS results in prostatic hyperplasia in the anterior and ventral lobes as well as increased levels of apoptosis throughout the prostate. Dysplastic foci were observed less frequently but were associated with the loss of E-cadherin staining and the loss of high molecular weight cytokeratin-positive basal epithelial cells. SSeCKS-null prostate tissues expressed significantly higher relative levels of AKT(poS473) compared with wild-type controls, suggesting that SSeCKS attenuates phosphatidylinositol-3-OH kinase signaling. The data suggest that SSeCKS-null mice have increased susceptibility for oncogenic transformation in the prostate.

Rb-dependent cellular senescence, multinucleation and susceptibility to oncogenic transformation through PKC scaffolding by SSeCKS/AKAP12

A subset of AKAPs (A Kinase Anchoring Proteins) regulate signaling and cytoskeletal pathways through the spaciotemporal scaffolding of multiple protein kinases (PK) such as PKC and PKA, and associations with the plasma membrane and the actin-based cytoskeleton. SSeCKS/Gravin/Akap12 expression is severely downregulated in many advanced cancers and exhibits tumor- and metastasis-suppressing activity. akap12-null (KO) mice develop prostatic hyperplasia with focal dysplasia, but the precise mechanism how Akap12 prevents oncogenic progression remains unclear. Here, we show that KO mouse embryonic fibroblasts (MEF) exhibit premature senescence marked by polyploidy and multinucleation, and by increased susceptibility to oncogenic transformation. Although p53 and Rb pathways are activated in the absence of Akap12, senescence is dependent on Rb. Senescence is driven by the activation of PKCα, which induces p16Ink4a/Rb through a MEK-dependent downregulation of Id1, and PKCδ, which downregulates Lats1/Warts, a mitotic exit network kinase required for cytokinesis. Our data strongly suggest that Akap12 controls Rb-mediated cell aging and oncogenic progression by directly scaffolding and attenuating PKCα/δ.

Pivotal Role of AKAP12 in the Regulation of Cellular Adhesion Dynamics: Control of Cytoskeletal Architecture, Cell Migration, and Mitogenic Signaling

Cellular dynamics are controlled by key signaling molecules such as cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). AKAP12/SSeCKS/Gravin (AKAP12) is a scaffold protein for PKA and PKC which controls actin-cytoskeleton reorganization in a spatiotemporal manner. AKAP12 also acts as a tumor suppressor which regulates cell-cycle progression and inhibits Src-mediated oncogenic signaling and cytoskeletal pathways. Reexpression of AKAP12 causes cell flattening, reorganization of the actin cytoskeleton, and the production of normalized focal adhesion structures. Downregulation of AKAP12 induces the formation of thickened, longitudinal stress fibers and the proliferation of adhesion complexes. AKAP12-null mouse embryonic fibroblasts exhibit hyperactivation of PKC, premature cellular senescence, and defects in cytokinesis, relating to the loss of PKC scaffolding activity by AKAP12. AKAP12-null mice exhibit increased cell senescence and increased susceptibility to carcinogen-induced oncogenesis. The paper describes the regulatory and scaffolding functions of AKAP12 and how it regulates cell adhesion, signaling, and oncogenic suppression.

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.

A transgenic mouse model for early prostate metastasis to lymph nodes

The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The individual genetic loss of the metastasis suppressor, SSeCKS/Gravin/AKAP12 or Rb, genes that are downregulated or deleted in human prostate cancer, results in prostatic hyperplasia. Here, we show that the combined loss of Akap12 and Rb results in prostatic intraepithelial neoplasia (PIN) that fails to progress to malignancy after 18 months. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes, marked by relatively differentiated tumor cells expressing markers of basal (p63, cytokeratin 14) and luminal (cytokeratin 8 and androgen receptor) epithelial cells, although none expressed the basal marker, cytokeratin 5. The finding that PIN lesions contain increased numbers of p63/AR-positive, cytokeratin 5-negative basal cells compared with WT or Akap12-/- prostate lobes suggests that these transitional cells may be the source of the lymph node metastases. Taken together, these data suggest that in the context of Rb loss, Akap12 suppresses the oncogenic proliferation and early metastatic spread of basal-luminal prostate tumor cells.

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 others expression. Our study implicates a role for SSeCKS in preventing of skin cancer progression possibly through negatively regulating FAK expression.

SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [1]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[BrafV600E] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naïve WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naïve KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21waf1 and p16ink4a, and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[BrafV600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

Abstract 3863: Transgenic model for early prostate metastasis to the lymph nodes.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The SSeCKS/Gravin/AKAP12 (“SSeCKS”) gene, previously shown to suppress prostate cancer metastasis upon re-expression ([1][1],[2][2]), as well as the tumor suppressor gene, Rb, are either transcriptionally downregulated or deleted in human prostate cancers ([3][3],[4][4]). Mouse models deficient in either SSeCKS or Rb exhibit prostatic hyperplasia ([5][5],[6][6]). Moreover, SSeCKS-null fibroblasts as well as SSeCKS-null prostates display Rb-dependent premature senescence markers ([7][7]). Methods: In order to determine whether the combined loss of AKAP12 and Rb in the prostate synergizes to induce oncogenic progression, Akap12-/- mice were crossed with Pb4-Cre;RbloxP/loxP mice to generate Akap12-/-;RbPE-/− progeny, with the Probasin-Cre passed only through males. Results: The combined loss of SSeCKS and Rb results in prostatic intraepithelial neoplasia (PIN) starting at 6 months of age that fails to progress to malignancy (adenocarcinoma) after 18 months. The PIN lesions were marked by increased Ki-67 proliferation of cytokeratin 8 (CK8), p63-negative luminal cells as well as p63-positive basal cells. Interestingly, these lesions also had increased numbers of androgen-receptor (AR)-positive, p63-positive, CK5-negative cells. There was evidence of reactive stroma including mural hyperplasia and inflammatory cell infiltration. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes (LN), marked by well-differentiated tumors cells expressing markers of basal (p63, CK14) and luminal (CK8 and AR) epithelial cells, although none expressed the basal marker, CK5. PCR-based tests for the deleted floxed Rb allele from laser capture microdissected LN lesions confirmed the prostatic origin of these metastatic cells. The LN lesions showed very limited inflammation, based on very few cells staining with the histiocyte marker, CD68. Conclusions: Taken together, these data suggest that in the context of Rb loss, SSeCKS suppresses the oncogenic proliferation and early metastatic spread of transitional, basal-luminal prostate tumor cells. Citation Format: Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, David W. Goodrich, Barbara A. Foster, Irwin H. Gelman. Transgenic model for early prostate metastasis to the lymph nodes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2013-3863 [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: #ref-7

Abstract C56: The combined loss of SSeCKS/Gravin/AKAP12 and Rb in the prostate promotes spontaneous lymph node metastases with basal-luminal transitional phenotypes

Abstract Introduction: Previous data indicated that the loss of SSeCKS/Gravin/AKAP12 (“SSeCKS”) or Rb correlates with prostate cancer (CaP) progression and that their deletion results in increased prostatic hyperplasia (1,2). SSeCKS re-expression in MAT-LyLu prostate cancer cells has little effect on the growth potential of primary-site tumors and on the ability of metastatic cells to colonize distal sites, yet it suppresses the growth of macrometastases by suppressing tumor-encoded vascular endothelial growth factor expression and neovascularization of the metastatic niche(3). We demonstrated recently that SSeCKS-null ( Akap12−/− ) mouse embryo fibroblasts (KO-MEF) suffer from an Rb-dependent premature senescence, and that the hyperplastic prostates in KO mice exhibit markers of increased senescence (4). Experimental Procedures: In order to determine whether SSeCKS and Rb act synergistically to promote CaP progression, KO mice were crossed to mice with prostate-specific conditional Rb deletion ( Pb-Cre;Rb fl/fl ). Results: Compared to controls (WT, Akap12−/− or Pb-Cre;Rb fl/fl mice), 100% of Akap12−/−;Pb-Cre;Rb fl/fl male mice showed evidence of low grade prostatic intraepithelial neoplasia (LG-PIN), with a smaller percentage (22%) displaying high grade (HG) PIN by 10 months of age. These lesions were marked by increased levels of cytoplasmic E-cadherin (compared to the cell-cell junctional staining in normal or hyperplastic luminal prostate layers) in LGPIN and by the loss of E-cadherin expression in HG-PIN. Unexpectedly, Akap12−/−;Pb-Cre;Rb fl/fl males showed increased numbers (1.8- to 2.5-fold) of p63-positive basal cells in all four lobes compared with control mouse prostates. Importantly, whereas control mice showed neither primary-site prostate cancer nor lymph node (LN) metastasis, >/80% of the Akap12−/−;Pb-Cre;Rb fl/fl males displayed metastases in pelvic or inguinal LN. These metastases did not stain for E-cadherin, but were positive for both basal (5 and 14) and luminal (8) cytokeratins, and low levels of androgen receptor. Foci within these lesions were positive for the basal marker, p63, and for the mesenchymal marker, vimentin. The LN lesions contained deletions of their floxed Rb loci, as shown by nested PCR assays, corroborating that they derived from the transgenic prostate cells. Conclusions: The combined loss of SSeCKS and Rb leads to early CaP initiation marked by increased basal cell proliferation in all four lobes, as well as the marked increase in LN metastasis of tumor cells with a basal-luminal transitional phenotype. These data strongly suggest that SSeCKS suppresses parameters of CaP initiation and metastasis. Citation Format: Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, Barbara Foster, Irwin H. Gelman. The combined loss of SSeCKS/Gravin/AKAP12 and Rb in the prostate promotes spontaneous lymph node metastases with basal-luminal transitional phenotypes [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C56.