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

Abstract

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.

Fig. 1. SSeCKS deficiency facilitates squamous papilloma formation

(A) Squamous papilloma incidence per mouse (percentage over time). (B) Papilloma multiplicity (average papilloma numbers/mouse). Variation between mice in either WT or KO groups was less than 15%. (C) Papilloma size in mm². Horizontal line, mean of 16 randomly selected lesions. *, P<0.001. (D) Representative images of skin papillomas (red arrows) in WT or KO mice after 28 weeks of treatment.

Fig. 2. SSeCKS deficiency promotes epidermal neoplasia

Pathologic analysis (H&E stained) of typical DMBA/TPA-induced (28 weeks) skin lesions from WT or KO mice. Note the limited SCC development in the WT lesion, which mostly contains benign hyperplastic epidermis. In contrast, most of the KO lesion contains SCC, including areas of more advanced, endophytic growth, containing moderately undifferentiated carcinoma. p, papilloma; t, SCC. Size bar, 50 μm.

Fig. 3. The expression of FAK is increased in SSeCKS-null mice

(A; upper panels) H&E-stained sections of 3-month-old WT and KO epidermal layers (typical thickness shown by double-head arrows). Size bar, 10 μm. Double-head arrow, representative epidermis. Lower panel, average thickness (in μm) of epidermal layers between WT and KO mice (10/group; 3 measurements/mouse). *, P<0.005. (B) Immunohistochemical staining for FAK (upper panels) or IgG control (bottom panel) of epidermal layers from 3-month-old WT or KO mice. (C) FAK IHC staining in SCC lesions in WT vs. KO mice. KO SCC lesions exhibit 2.44-fold (+/− 0.62) higher FAK levels than WT SCC (mean staining levels in 5 independent fields for each mouse strain, as described in Materials and Methods. P<0.02). Immunoblot analysis for SSeCKS, FAK or GAPDH protein in lysates of SSeCKS+/+ (WT) or SSeCKS−/− MEF (D), or FAK+/+ or FAK−/− MEF (E), or in WT MEF treated with control (scrambled)-, mAKAP12 (SSeCKS)-, or FAK-siRNA (F). The blots in panels D–F are typical of three independent experiments. (G) IHC analysis of untreated skin of WT vs. KO mice stained for phospho-ERK1/2, phospho-JNK/SAPK or control IgG. (H) IHC staining of normal human skin vs. human SCC lesions for SSeCKS/Gravin/AKAP12. The 3-fold lower SSeCKS staining in SCC vs. normal skin was found in 10 independent tissue sets (not shown).