Advances in Brief Suppression of Type I Interferon Signaling Proteins Is an Early Event in Squamous Skin Carcinogenesis

Purpose: IFN-based therapy has been shown to be active in the treatment of squamous cell carcinoma (SCC) of the skin, the most aggressive form of non-melanoma skin cancer. Based largely on this activity, we began programmatically examining the expression of IFN-stimulated gene factor 3 (ISGF-3) proteins (signal transducers and activators of transcription 1 / , signal transducers and activators of transcription 2, and p48), which are important mediators of IFNsignaling, in skin premalignancy and SCC. Our previous preliminary studies suggested suppression of some or all of the ISGF-3 proteins in skin SCC. Experimental Design: To determine the timing of the suppression of IFNsignaling proteins in squamous skin carcinogenesis, we have now compared ISGF-3 expression by immunohistochemical staining in biopsies of actinic keratosis, a form of skin premalignancy, and matched normal skin. Results: We observed a significant decrease in expression of one or more ISGF-3 proteins in 76% of patients with actinic keratosis (19 of 25 patients). In addition, we found a suppression of one or more ISGF-3 proteins in 67% of skin SCC patients tested (12 of 18 patients), confirming our previous observations. Conclusions: These data have led to the hypothesis that the suppressed expression of ISGF-3 proteins and consequent reduction in responsiveness to endogenous IFN likely are an early event in skin carcinogenesis. Introduction Non-melanoma skin cancer is the single most commonly diagnosed cancer in the Caucasian population, with 1,000,000 new cases reported in the United States each year (1). Of the two most common forms of skin cancer (SCC and basal cell carcinoma), SCC is clinically the most aggressive, accounting for most of the non-melanoma skin cancer deaths, and has been increasing in incidence since the 1960s, with especially increased rates in recent years. Currently, successful treatment of locally advanced skin SCC involves surgery and radiotherapy. However, aggressive SCC lesions recur at a high rate. AK is a premalignant condition, or IEN, of the skin characterized by keratinocytic atypia, which lies along the causal pathway between normal skin and SCC (2, 3). Thus, genetic or epigenetic alterations leading to AK formation can be considered early events in skin carcinogenesis. IFNs are cytokines that regulate proliferation, differentiation, and immune function (4). Type I IFNs (IFNand ) both bind to cell surface receptors composed of two distinct subunits: IFNreceptor 1 and IFNreceptor 2. The receptors activate associated kinases, Janus kinase 1 and tyk2, members of the Janus kinase family of receptor-associated tyrosine kinases. These kinases then phosphorylate several different STATs, members of a family of latent cytoplasmic transcription factors, resulting in their translocation to the nucleus, where they can modulate the transcription of several genes (5). Although they are not the only STATs activated by type I IFNs, STAT1 and STAT2 are likely the most important STATs mediating type I IFN effects and are phosphorylated on tyrosines 701 and 690, respectively (4). The phosphorylated (activated) STAT1 and STAT2 proteins complex with a third protein, p48, to form the ISGF-3 transcription factor (6). After translocation to the nucleus, the p48 component of the ISGF-3 binds to DNA sequences called IFN-stimulated response elements, found in the promoters of most type I IFN-responsive genes (6). IFN-based therapy has been used successfully for the treatment of several malignancies (7), including SCC of the skin, with greater activity in premalignancies and locally advanced SCC than in metastatic disease (8, 9). Based on this activity and the established roles of ISGF-3 proteins in mediating IFN effects, we began to programmatically study the expression pattern of type I IFN signaling proteins in skin SCC. We previously reported our findings of a suppressed expression of some or all of these proteins in patient samples of aggressive skin SCC relative to adjacent NM epithelium for the majority of a limited number (n 12) of patients (10). In the present study, we compared ISGF-3 expression in AKs with that in matched Received 1/9/01; revised 3/11/02; accepted 3/23/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by National Cancer Institute Grants 5 R29 CA78560 and 1 P01 CA88233 and National Institute of Environmental Health Sciences Grant 5 P30 ES07784. 2 To whom requests for reprints should be addressed, at Department of Clinical Cancer Prevention, The University of Texas M. D. Anderson Cancer Center, Box 236, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-0627; Fax: (713) 792-0628; E-mail: [email protected]. 3 The abbreviations used are: SCC, squamous cell carcinoma; AK, actinic keratosis; IEN, intraepithelial neoplasia; STAT, signal transducers and activators of transcription; ISGF-3, IFN-stimulated gene factor-3; NM, nonmalignant; RA, retinoic acid. 2067 Vol. 8, 2067–2072, July 2002 Clinical Cancer Research Research. on September 22, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from normal skin biopsies to determine whether altered ISGF-3 expression is potentially an early event in skin carcinogenesis. This determination can advance our molecular understanding of the early events in skin cancer development, which is crucial for chemoprevention in this setting (3, 11). We also extended our earlier preliminary observation of ISGF-3 protein suppression in aggressive skin SCC to a larger number of patient samples. Materials and Methods Surgical Specimens. Surgical specimens of AK were obtained from sun-exposed areas of skin from Caucasian patients (ages range, 46–92 years). AK specimens were removed surgically or by shave biopsy, and matched normal skin samples were removed from a distant non-sun-exposed site by punch biopsy. Baseline surgical specimens of aggressive skin SCC came from a Phase III trial of IFN-based therapy (National Cancer Institute CA88233). Aggressive tumors were defined as having met one or more of the following criteria: (a) size 2 cm diameter; (b) perineural invasion; (c) nodal involvement; or (d) deep structure (e.g., muscle) involvement. SCC specimens were selected to include tumor and adjacent NM tissue. All specimens were processed by a routine fixation in 10% neutral Bouin’s fixative and embedded in paraffin. All specimens were cut to 4M sections and attached to lysine-coated slides. Immunostaining of Paraffin Sections. Handling of sections, antigen retrieval, antibodies, and staining conditions were as described previously (10), with the addition of a pancytokeratin antibody (Sigma) that served as a positive staining control. For each antibody, the entire set of AK and matched normal or SCC specimens was processed and immunostained at the same time. The specificity of the antibodies was further verified by Western blotting (data not shown). To control for nonspecific binding of primary antibodies, duplicate samples were incubated with the STAT1 / , STAT2, and p48 antibodies Fig. 1 ISGF-3 proteins are suppressed in AK relative to normal skin to a degree similar to aggressive SCC. Thin sections (4M) of paraffin-embedded biopsy specimens were immunohistochemically stained with antibodies for the indicated proteins. Normal Skin indicates skin biopsy material taken from a distant nonsun-exposed site. Actinic Keratosis is from sun-exposed skin from the same patient. SCC indicates biopsy material from an aggressive skin SCC taken from a different patient than the samples in the other columns. Pan-cyto indicates staining with a pancytokeratin antibody control. All images were photographed at identical magnification ( 200). 2068 Interferon Signaling in Skin Carcinogenesis Research. on September 22, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from along with either the corresponding immunizing peptides or an irrelevant peptide. In all cases, the immunizing peptide and not the irrelevant peptide blocked staining (Ref. 10; data not shown). Duplicate control specimens receiving only second antibody did not stain. Slides were photographed at 200 magnification under oil immersion with a Nikon digital camera. Densitometric Quantitation of Protein Expression. Digital images of normal skin, adjacent NM skin, AK, and SCC were captured under identical light intensity, exposure time, and camera settings with a Photometrics Quantix charge-coupled device camera at 200 magnification, using a blue filter, essentially as described previously (10). Image files were analyzed with IPLabs quantitation software. Three different fields were chosen randomly for normal skin, adjacent NM skin, AK, and SCC and averaged for each specimen. Note that counterstaining was omitted to allow densitometric quantitation of only the brown color of the converted diethyl amino benzene peroxidase substrate.