Loss of the SSeCKS/Gravin/AKAP12 gene results in prostatic hyperplasia

Abstract

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.

Figure 1

Targeted disruption of the ssecks/gravin/akap12 gene. A, a partial map of the ssecks locus and diagrams showing the gene-targeting strategy. Filled boxes, exons 2 and 3 (Ex2 and Ex3, respectively). An upstream 6.5-Kb fragment was amplified with primers sc4F/R, spliced upstream of the lacZ/neo^R cassette in pTCT5.2, followed by the insertion of the downstream XbaI/SpeI 3.0-Kb ssecks fragment, as described in Materials and Methods. ES clones with the targeting vector inserted into an ssecks genomic locus were confirmed by Southern blotting on EcoRI cut genomic DNA using lacZ and 3′-exon 2 probes (hatched boxes), as well as long-run PCR analysis using primers NeoF and sc19R (data not shown). B, PCR analysis of mouse tail DNA showing ssecks WT or targeted (mt) alleles in WT (+/+), heterozygous (+/−) and homozygous (−/−) mice. C, Southern blot analysis of XbaI-cut genomic DNA from WT (+/+), heterozygotes (+/−), or KO (−/−) mice probed with a 480-bp fragment mapping downstream of exon 3 and outside the targeting vector (A). Whereas the 3-kb XbaI fragment is diagnostic of the WT allele, the 15-kb XbaI fragment is diagnostic of the recombinant KO allele (A).

Figure 2

SSeCKS-null mice exhibit increased prostate weight, cellularity, cell proliferation, and apoptotic index. A, quantification of hypercellularity of anterior (AP), dorsal (DP), lateral (LP), and ventral (VP) prostatic lobes in WT (white columns) versus KO mice (black columns). H&E-stained paraffin sections of individually dissected lobes from at least six age-matched WT or KO mice were analyzed for cellularity: the number of cells per duct were counted in three equally sized ducts for each lobe. Columns, mean; bars, SE; *, P < 0.01; **, P < 0.05. B, the average total weight of prostates from WT versus KO mice. Prostates from at least six mice per age group were dissected, and the prostate weights (normalized to total body weight) were determined. Columns, mean; bars, SE; *, P <0.01 (C). Proliferation indices were determined based on Ki67 staining of paraffin sections of dissected prostate lobes from three 15-mo-old WT or KO mice. At least three equally sized ducts/lobe to analyze the percentage of Ki67-positive cells/lobe. Columns, mean; bars, SE; *, P < 0.001; **, P < 0.01. D, the average level of apoptosis was determined on the same tissue samples as in panel C using immunohistochemical staining for TUNEL as described in Materials and Methods. Columns, mean; bars, SE; *, P < 0.005.

Figure 3

Hyperplasic changes in the prostates of SSeCKS-null mice. H&E-stained sections of anterior, dorsal, lateral, and ventral prostate lobes derived from 15-mo-old WT or KO mice. Scale bar, 50 μm.

Figure 4

Hyperactivation of AKT in SSeCKS-null prostates. A, immunohistochemical staining of Akt^poS473 (or control IgG) in paraffin sections of AP lobes from 15-mo-old WT or KO mice. This is a typically result of at least three independent assays. B, Western blot analysis showing increased relative Akt^poS473 levels in KO versus WT AP lobes. SSeCKS protein levels are shown to confirm the KO status of these tissues, and GAPDH levels are shown as protein loading controls. These data are consistent in at least two independent prostate isolations. Cont., control.

Figure 5

Loss of cell-cell junctions and basal epithelial cells in SSeCKS-null AP lobes. Immunohistochemical analysis of E-cadherin (A) or high molecular weight cytokeratins (B) in the AP paraffin sections from 3-mo-old WT or KO mice. The portion of the inset boxed region in the middle of A is magnified at right. Scale bars, 50 μm.