Stage-specific disruption of Stat3 demonstrates a direct requirement during both the initiation and promotion stages of mouse skin tumorigenesis

Abstract

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss-of-function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER(T2) x Stat3(fl/fl)) that show epidermal-specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by intraperitoneal injection) led to inhibition of tumor growth compared with tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Fig. 1.

Induction of Stat3 deficiency in epidermis of K5.Cre-ER^T2 × Stat3^fl/fl mice by application of TM. (A) Mice were segregated by genotype into four groups and treated as shown. (B) Western blot analysis and quantification of Stat3 level in inducible Stat3-deficient mice treated with TM (Group 1) and control (Groups 2–4) mice. (C) Immunohistochemical staining for Stat3 in skin sections from inducible Stat3-deficient mice treated with ethanol or TM. (D) Western blot analysis of Stat3 and phosphorylated Stat3 levels in relation to the levels of Bcl-xL, Cyclin D1, Cyclin E and c-Myc in inducible Stat3-deficient mice treated with TM (Group 1) and the control groups (Groups 2–4).

Fig. 2.

Temporal disruption of Stat3 results in increased apoptosis and decreased cell proliferation in the epidermis of inducible Stat3-deficient mice. (A) DMBA-induced apoptosis in the epidermis of inducible Stat3-deficient mice. Groups of mice (n = 3) were treated with TM or ethanol as indicated (Figure 1A) for five consecutive days, then 24 h later they received a single topical treatment of 25 nmol DMBA (black bars) or acetone (white bars). Mice were then killed 24 h later and skin sections were prepared for analysis. (B) TPA-induced cell proliferation in the epidermis of inducible Stat3-deficient mice. Groups of mice (n = 3) were treated with TM or ethanol as indicated (Figure 1A) for five consecutive days. Twenty-four hours after the last treatment of TM, they were treated with 6.8 nmol TPA (black bars) or acetone (white bars) once and were killed 24 h later. BrdU (100 μg/g body wt) was injected 30 min prior to killing. (Group 1, inducible Stat3-deficient mice treated with TM; Group 2, inducible Stat3-deficient mice treated with ethanol; Group 3, Cre⁻ mice treated with TM and Group 4, Cre⁻ treated with ethanol). *P < 0.01 by Mann–Whitney U-test.

Fig. 3.

Effect of Stat3 disruption on the initiation stage of two-stage skin carcinogenesis. (A–C) Groups of mice were treated with 25 nmol of DMBA and after 4 weeks received twice-weekly applications of 6.8 nmol of TPA for the duration of the experiment. TM or ethanol was applied topically for five consecutive days either before initiation with DMBA (Groups 1–4) or after initiation with DMBA (Group 5) as shown in (A). (B) Percentage of mice with papillomas and (C) average number of papillomas per mouse [Group 1, inducible Stat3-deficient mice treated with TM (filled circles); Group 2, inducible Stat3-deficient mice treated with ethanol (open circles); Group 3, Cre⁻ mice treated with TM (filled squares); Group 4, Cre⁻ mice treated with ethanol (open squares) and Group 5, inducible Stat3-deficient mice treated with TM after DMBA initiation (filled triangles)].

Fig. 4.

Effect of Stat3 disruption on the tumor promotion stage of two-stage skin carcinogenesis. (A–C) Groups of mice were treated with 100 nmol of DMBA and after 2 weeks received twice-weekly applications of 6.8 nmol of TPA for the duration of the experiment. TM or ethanol was applied topically for five consecutive days and repeated every 2 weeks and then stopped after 16 weeks. (A) Experimental protocol for disruption of Stat3 during the promotion stage. (B) Percentage of mice with papillomas and (C) average number of papillomas per mouse [Group 1, inducible Stat3-deficient mice treated with TM (filled circles); Group 2, inducible Stat3-deficient mice treated with ethanol (open circles); Group 3, Cre⁻ mice treated with TM (filled squares) and Group 4, Cre⁻ mice treated with ethanol (open squares)].

Fig. 5.

Effects of TM-induced Stat3 deficiency on growth of skin tumors. (A–B) Groups of mice were treated with 25 nmol of DMBA and 2 weeks later received twice-weekly applications of 6.8 nmol of TPA for 26 weeks. After 2 weeks without TPA treatment, the mice were then treated with TM by i.p. injection for five consecutive days on alternate weeks over the 4 week period. (A) Experimental protocol for Stat3 disruption in skin tumors. (B) Average size of skin tumors per group [Group 1, inducible Stat3-deficient mice treated with TM (filled circles); Group 2, inducible Stat3-deficient mice treated with ethanol (open circles); Group 3, Cre⁻ mice treated with TM (filled squares) and Group 4, Cre⁻ mice treated with ethanol (open squares)].