Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression

Abstract

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

Figure 1.

Deletion of FAK from mouse skin blocks papilloma formation. (A) fak excision in ES cells following introduction of Cre recombinase. Cre was introduced by electroporation of a pMC1-Cre plasmid, and protein extracts were harvested and subjected to Western blotting with an FAK mAb. (Lanes 1-3) No Cre recombinase. (Lanes 4-6) Cre recombinase. (B) Proteins were harvested from spleen, kidney, and skin of K14CreER^T2/FAK^flox/flox mice either treated with 4-OHT or with vehicle alone. Extracts were blotted onto nitrocellulose and probed with an anti-FAK mAB (top panel) or an anti-tubulin mAB (lower panel). (C) Immunohistochemical staining of paraffin-embedded skin sections from K14CreER^T2/FAK^flox/flox mice treated with either 4-OHT (right panel) or vehicle alone (left panel). (E) Epidermis; (HF) hair follicles. Deletion of fak in epidermal keratinocytes in vivo inhibits papilloma formation during chemical carcinogenesis. Mice were either treated with 4-OHT (♦) or vehicle alone (□) and subjected to DMBA and TPA treatment as described. Graphs indicate percent of mice acquiring papillomas over time (D) and average numbers of papillomas recorded per mouse over treatment time (E). Graphs represent K14CreER^T2/FAK^flox/flox mice.

Figure 2.

Deletion of fak from papillomas blocks malignant progression to SCC. (A) Mice either K14CreER^T2/FAK^flox/flox (left panel) or FAK^flox/flox (right panel) were treated with vehicle alone or with 4-OHT (+4-OHT) at 15 wk after the majority of papillomas had formed. (B) Immunohistochemical anti-FAK staining of paraffin-embedded sections of papillomas from K14CreER^T2/FAK^flox/flox mice treated with vehicle alone (left panel) and with 4-OHT (right panel). (C) Papilloma to carcinoma conversion frequency of 4-OHT-treated and untreated K14CreER^T2/FAK^flox/flox mice (1 and 2) and 4-OHT-treated FAK^flox/flox mice (3). Conversion frequency is determined by calculating the number of carcinomas recorded per total number of papillomas for each study group. (D) Anti-FAK immunoblot of proteins extracted from SCCs derived from untreated (lanes 1-6) or 4-OHT-treated (lanes 7,8) K14CreER^T2/FAK^flox/flox mice.

Figure 3.

Deletion of FAK from keratinocytes causes reduced migration and increased cell death. (A) Confocal images show anti-FAK staining of peripheral structures in untreated (-4-OHT) or treated (+4-OHT) keratinocytes derived from K14CreER^T2/FAK^flox/flox mice. Arrows indicate FAK staining in a minority of treated keratinocytes. Images were taken following 48 h of 4-OHT treatment. (B) Phase-contrast images show in vitro wound-repair assays of untreated (-4-OHT) or treated (+4-OHT) keratinocytes derived from K14CreER^T2/FAK^flox/flox mice at 0 h and 48 h after the wound was made in the monolayer. Arrows indicate areas of the monolayer at the edges of the wound after 48 h. (C) Migration rates of keratinocytes derived from FAK^flox/flox mice (1) and K14CreER^T2/FAK^flox/flox mice (2), treated with 4-OHT for 48 h, were determined by time-lapse cell tracking and migration rate software analysis expressed as average speed in microns per second. (D,E) Cell death of untreated (-4-OHT) or treated (+4-OHT) keratinocytes derived from K14CreER^T2/FAK^flox/flox mice was judged by the percentage of cells with sub-2n DNA content by FACS analysis; 24 h and 48 h refers to the time of sample collection after wounding.

Figure 4.

(A) Sections of wounded skin from 4-OHT-treated (+4-OHT) or untreated (-4-OHT) K14CreER^T2/FAK^flox/flox mice were stained with H&E to examine re-epithelialization at day 0 (upper panels) and day 7 (lower panels) post-injury. Arrows indicate the leading edges of migrating epidermis. (B) Immunohistochemical anti-activated caspase-3 staining of sections of either 4-OHT-treated (+4-OHT) or untreated (-4-OHT) skin (upper and middle panels) or papillomas (lower panels) from K14CreER^T2/FAK^flox/flox mice. Images taken at either low (upper panels) or high (middle and lower panels) magnification. (HF) Hair follicles.