Here we report that epidermal keratinocytes in psoriatic lesions are characterized by activated Stat3. Transgenic mice with keratinocytes expressing a constitutively active Stat3 (K5.Stat3C mice) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis. Keratinocytes from K5.Stat3C mice show upregulation of several molecules linked to the pathogenesis of psoriasis. In addition, the development of psoriatic lesions in K5.Stat3C mice requires cooperation between Stat3 activation in keratinocytes and activated T cells. Finally, abrogation of Stat3 function by a decoy oligonucleotide inhibits the onset and reverses established psoriatic lesions in K5.Stat3C mice. Thus, targeting Stat3 may be potentially therapeutic in the treatment of psoriasis.

For more than 60 years, the chemical induction of tumors in mouse skin has been used to study mechanisms of epithelial carcinogenesis and evaluate modifying factors. In the traditional two-stage skin carcinogenesis model, initiation is accomplished by the application of a subcarcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor promoting agent. The initiation protocol can be completed within 1–3 hours depending on the number of mice used, while the promotion phase requires twice weekly treatments (1–2 hours) and once weekly tumor palpation (1–2 hours) for the duration of the study. A highly reproducible papilloma burden is expected within 10–20 weeks with progression of a portion of the tumors to squamous cell carcinomas within 20–50 weeks. In contrast to complete skin carcinogenesis, the two-stage model allows for greater yield of premalignant lesions as well as separation of the initiation and promotion phases.

Loss of tuberin, the product of TSC2 gene, increases mammalian target of rapamycin (mTOR) signaling, promoting cell growth and tumor development. However, in cells expressing tuberin, it is not known how repression of mTOR signaling is relieved to activate this pathway in response to growth factors and how hamartin participates in this process. We show that hamartin colocalizes with hypophosphorylated tuberin at the membrane, where tuberin exerts its GTPase-activating protein (GAP) activity to repress Rheb signaling. In response to growth signals, tuberin is phosphorylated by AKT and translocates to the cytosol, relieving Rheb repression. Phosphorylation of tuberin at serines 939 and 981 does not alter its intrinsic GAP activity toward Rheb but partitions tuberin to the cytosol, where it is bound by 14-3-3 proteins. Thus, tuberin bound by 14-3-3 in response to AKT phosphorylation is sequestered away from its membrane-bound activation partner (hamartin) and its target GTPase (Rheb) to relieve the growth inhibitory effects of this tumor suppressor.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

It has long been known that excessive mitotic activity due to H-Ras can block keratinocyte differentiation and cause skin cancer. It is not clear whether there are any innate surveillants that are able to ensure that keratinocytes undergo terminal differentiation, preventing the disease. IKKalpha induces keratinocyte terminal differentiation, and its downregulation promotes skin tumor development. However, its intrinsic function in skin cancer is unknown. Here, we found that mice with IKKalpha deletion in keratinocytes develop a thickened epidermis and spontaneous squamous cell-like carcinomas. Inactivation of epidermal growth factor receptor (EGFR) or reintroduction of IKKalpha inhibits excessive mitosis, induces terminal differentiation, and prevents skin cancer through repressing an EGFR-driven autocrine loop. Thus, IKKalpha serves as an innate surveillant.

In the present study, we have investigated the possible role of signal transducers and activators of transcription (STATs), particularly Stat3, in mouse skin tumor promotion and multistage carcinogenesis. Stat1, Stat3, and Stat5 were activated in mouse epidermis after treatment with different classes of tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and chrysarobin. In addition, Stat1, Stat3, and Stat5 were constitutively activated in skin tumors generated by the two-stage carcinogenesis regimen using 7,12-dimethylbenz(a)anthracene as initiator and TPA as promoter. Several approaches were used to examine the possible role of epidermal growth factor receptor (EGFR) in modulating Stat3 activity during tumor promotion. In primary cultures of mouse keratinocytes, addition of exogenous EGF led to activation of Stat3 as shown by an elevation in tyrosine phosphorylation and nuclear translocation. In epidermis of transgenic mice expressing transforming growth factor ␣ under control of the keratin 14 promoter, Stat3 was constitutively activated. Abrogation of EGFR function in mouse epidermis using an EGFR kinase inhibitor or by overexpressing a dominant negative form of EGFR led to a reduction in Stat3 activation in response to TPA treatment. Immunoprecipitation analyses using lysates from TPA-treated epidermis and skin papillomas showed enhanced interaction between the EGFR and Stat3. Finally, Stat3 deficiency in mouse epidermis significantly reduced the proliferative response after TPA treatment. Collectively, the current results suggest that Stat3 activation may be a critical event during mouse skin tumor promotion, possibly through regulation of keratinocyte proliferation. In addition, Stat3 activation in tumor promoter-treated epidermis and in skin papillomas may occur, at least in part, via interaction with and phosphorylation by the EGFR. Finally, constitutive activation of Stat3 in both papillomas and squamous cell carcinomas suggest a role in both the development of autonomous growth and the progression of epithelial tumors in mouse skin.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

Gemcitabine (Gemzar®) is the first line treatment for pancreatic cancer and often used in combination therapy for non-small cell lung, ovarian, and metastatic breast cancers. Although extremely toxic to a variety of tumor cells in culture, the clinical outcome of gemcitabine treatment still needs improvement. In the present study, a new gemcitabine nanoparticle formulation was developed by incorporating a previously reported stearic acid amide derivative of gemcitabine into nanoparticles prepared from lecithin/glyceryl monostearate-in-water emulsions. The stearoyl gemcitabine nanoparticles were cytotoxic to tumor cells in culture, although it took a longer time for the gemcitabine in the nanoparticles to kill tumor cells than for free gemcitabine. In mice with pre-established model mouse or human tumors, the stearoyl gemcitabine nanoparticles were significantly more effective than free gemcitabine in controlling the tumor growth. PEGylation of the gemcitabine nanoparticles with polyethylene glycol (2000) prolonged the circulation of the nanoparticles in blood and increased the accumulation of the nanoparticles in tumor tissues (> 6-fold), but the PEGylated and un-PEGylated gemcitabine nanoparticles showed similar anti-tumor activity in mice. Nevertheless, the nanoparticle formulation was critical for the stearoyl gemcitabine to show a strong anti-tumor activity. It is concluded that for the gemcitabine derivate-containing nanoparticles, cytotoxicity data in culture may not be used to predict their in vivo anti-tumor activity, and this novel gemcitabine nanoparticle formulation has the potential to improve the clinical outcome of gemcitabine treatment.