Stat3 as a Therapeutic Target for the Treatment of Psoriasis: A Clinical Feasibility Study with STA-21, a Stat3 Inhibitor  Ken Miyoshi, Mikiro Takaishi, Kimiko Nakajima, Mitsunori Ikeda, Takashi Kanda, Masahito Tarutani, Tatsuo Iiyama, Naoki Asao, John DiGiovanni, Shigetoshi Sano  Journal of Investigative Dermatology  Volume 131, Issue 1, Pages 108-117 (January 2011) DOI: 10.1038/jid.2010.255 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Activated Stat3 in psoriatic lesional skin and increased mRNA levels of IL-20 subfamily cytokines and HB-EGF. (a) Immunofluorescence staining with an anti-Stat3 antibody showed activated Stat3 in epidermal keratinocytes of psoriatic lesions, whereas no Stat3 was detected in the uninvolved skin from psoriasis patients or from normal control skin. Sections were counterstained with 46-diamidino-2-phenyl indole (DAPI). H&E, hematoxylin-eosin staining. Bar=250μm. (b) Expression of IL-22, IL-20, IL-19, IL-4, and heparin-binding EGF-like growth factor (HB-EGF) mRNAs in lesional skin relative to uninvolved skin from patients with psoriasis (n=5). IL-20 subfamily cytokines and HB-EGF mRNAs were increased in the lesional skin, but IL-4 mRNA was not increased. Journal of Investigative Dermatology 2011 131, 108-117DOI: (10.1038/jid.2010.255) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 STA-21 inhibits Stat3 signaling in NHK. (a) Pretreatment of normal human keratinocyte (NHK) in culture with 20μM STA-21 for 1hour before stimulation with EGF, IL-6, or IL-22 (100ngml-1 for 30minutes). Immunostaining with an anti-Stat3 antibody showed that EGF, IL-6, or IL-22 induced nuclear translocation of Stat3, which was inhibited by STA-21. Bar=50μm. (b) STA-21 inhibited NHK proliferation in a dose-dependent manner measured using the MTS assay. Data are reported as mean relative cell numbers ±SD. DMSO (0.2%) is the vehicle control. STA-21 concentrations at 0, 2.5, 5, 10, 20, and 40 are indicated by lines in black, blue, green, purple, red, and orange, respectively. A representative result from four independent experiments is shown. (c) Cytotoxicity assay of STA-21 in vitro. Lactate dehydrogenase (LDH) is measured using the supernatants of NHK cultured for 24hours in the presence of STA-21 at the indicated concentrations. NHK in DMSO (0.2%) alone and freeze–thaw control are set at 0 and 100% of LDH release, respectively. Data are means±SD from triplicate cultures. (d) Downregulation of c-Myc and cyclin D1 mRNA in NHK by STA-21 (20μM). Relative transcript levels measured by real-time RT-PCR of the relative expression from NHK cultured in the presence of STA-21 for 24hours (shaded bars) and for 48hours (dotted bars) compared with untreated cells (black bars) are shown. A representative result from three independent experiments is shown. Journal of Investigative Dermatology 2011 131, 108-117DOI: (10.1038/jid.2010.255) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 STA-21 induces keratinocyte differentiation markers in vitro. (a) Morphology of normal human keratinocyte (NHK) cultured 24hours in the presence of STA-21 (10μM), high calcium (1.2mM), or calcitoriol (10-7M). Cells became flattened and large when treated with STA-21, similar to cells treated with calcium or calcitoriol. Bar=250μm. (b) Increases in mRNA levels of keratinocyte differentiation molecules, involucrin, transglutaminase 1 (TGase 1), and keratin 10, but not keratin 14. NHKs were treated with STA-21 for 24hours at concentrations of 0, 2.5, 5, 10, and 20μM (stepwise increments indicated by triangles). Relative mRNA expression of treated versus untreated cells is shown. A representative result from three independent experiments is shown. (c) Western blotting of NHK cells treated with STA-21 (20μM), high calcium (1.2mM), or calcitoriol (10-7M) for 48hours. STA-21 increased involucrin and transglutaminase proteins up to 1.5- and 7.6-fold, respectively, compared to the DMSO-treated control. In contrast, STA-21 decreased c-Myc and cyclin D1 proteins to 0.5- and 0.4-fold, respectively. (d) Downregulation of transcriptional levels of K10 and involucrin by IL-22 and possible involvement of Stat3. Stimulation of cultured NHKs with IL-22 (200ngml-1) for 24hours reduced the levels of involucrin (black bars) and K10 (shaded bars) mRNAs to 41 and 44% of the basal level, respectively. Simultaneous treatment with STA-21 (20μM) overcomes the inhibitory effect of IL-22 on the expression of both genes. Journal of Investigative Dermatology 2011 131, 108-117DOI: (10.1038/jid.2010.255) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 A decrease in Stat3 nuclear localization of mouse epidermis following topical treatment with STA-21. (a) Amount of nuclear-located Stat3. The amount of nuclear-localized Stat3 in epidermis is constitutively increased in K5.Stat3C mice, whereas that in wild-type mice is virtually absent. Tape stripping immediately enhances the Stat3 translocation, which is markedly inhibited by pretreatment with STA-21 (20μg) at 1hour before tape stripping. Normal mice (n=3), K5.Stat3C mice untreated (n=3), tape-stripped (TS) acetone control (n=3), and STA-21 pretreated (n=3). Epidermal keratinocytes positive for nuclear Stat3 staining are counted in three nonoverlapped fields per mouse. Data are mean number of cells positive for nuclear Stat3 ±SD. **P<0.01, ***P<0.001. (b) Immunohistochemical staining with anti-Stat3 of tape-stripped K5.Stat3C mouse skins pretreated 1hour earlier with acetone (top) and STA-21 (bottom). Lines, epidermis–dermis border. Bar=50μm. Journal of Investigative Dermatology 2011 131, 108-117DOI: (10.1038/jid.2010.255) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Topical treatment with STA-21 inhibits the generation of psorisiform lesions in K5.Stat3C mice. (a) STA-21 inhibited an early 12-O-tetradecanoylphorbol-13-acetate (TPA) response of the ear skin in K5.Stat3C mice. Ear skins of transgenic mice topically treated at days 1 and 3 with 0.64nmol TPA or acetone alone were sampled at day 4. Although TPA induced epidermal hyperplasia (black bars), treatment with STA-21 (10μg) for 3 consecutive days significantly reduced the TPA-induced epidermal thickness (red bars). Results are shown as mean thickness (μm) ±SD, ***P<0.001, n=6 and 5 (untreated and STA-21 treated, respectively). (b) Representative histological features of STA-21-treated ear skin. TPA-induced histological changes in the ear skin of K5.Stat3C mice (left panel) include epidermal hyperplasia, hyperkeratosis, dermal infiltrates (arrows), capillary proliferation, and dilation (arrowheads). STA-21 treatment markedly diminished those changes (right panel). H&E, hematoxylin-eosin staining. Bar=100μm. (c) STA-21 inhibited the development of bona fide psoriasiform lesions on the dorsal skin. Topical TPA treatment (3.4nmol) three times a week induced psoriasiform lesions on the back (upper right), with psoriasis-like histological changes (lower right). Application of STA-21 (20μg) three times a week to the TPA-treated area (depicted by circles) completely inhibited the generation of psoriasiform lesions macro- and microscopically (right panels). Bar=300μm. (d) Assessment of epidermal thickness. Black bar, vehicle controls (n=6); red bar, STA-21 treated (n=5). Data are reported as mean thickness (μm) ±SD, ***P<0.0001. Journal of Investigative Dermatology 2011 131, 108-117DOI: (10.1038/jid.2010.255) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Efficacy of topical treatment with STA-21 on psoriatic lesions. (a) Psoriatic lesions of two patients during the course (patient 1, a 56-year-old man; patient 2, a 83-year-old man). Improvement of psoriasis lesions following application of STA-21 on the indicated days compared with vehicle controls (Vaseline). (b) Sum of clinical scores of baseline, 1 week, and 2 weeks of lesions from all eight patients allocated in this study. Black line, Vaseline control; red line, STA-21 treated. Five patients (patients 1–5), who showed >30% difference in the score decline after 2 weeks treatment with STA-21 compared with vehicle control, are considered to be responders based on the scores. Δ% Score decline in patients 1–8: 50, 38, 31, 35, 63, 0, 0, and 14, respectively. (c) Summary data for the mean of clinical score across all eight patients. Mean±SD percent of the initial score is shown. Compared with vehicle controls (black line), STA-21-treated lesions (red line) showed clinical improvement. *P=0.026 (<0.05) and 0.056 at days 7 and 14, respectively. Journal of Investigative Dermatology 2011 131, 108-117DOI: (10.1038/jid.2010.255) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions