Inducible cutaneous inflammation reveals a protumorigenic role for keratinocyte CXCR2 in skin carcinogenesis.

Transgenic mice that overexpress PKCalpha in the epidermis (K5-PKCalpha mice) exhibit acute CXCR2-mediated intraepidermal neutrophilic inflammation and a strong epidermal hyperplasia in response to application of 12-O-tetradecanoylphorbol-13-acetate (TPA). We now show that hyperplasia is independent of infiltrating neutrophils. Furthermore, when K5-PKCalpha mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) and promoted with a low dose of TPA, 58% of K5-PKCalpha mice developed skin papillomas that progressed to carcinoma, whereas wild-type mice did not develop tumors. We confirmed that CXCR2 is expressed by keratinocytes and showed that transformation by oncogenic ras (a hallmark of DMBA initiation) or TPA exposure induced all CXCR2 ligands. Ras induction of CXCR2 ligands was mediated by autocrine activation of epidermal growth factor receptor and nuclear factor-kappaB, and potentiated by PKCalpha. Oncogenic ras also induced CXCR2 ligands in keratinocytes genetically ablated for CXCR2. However, ras transformed CXCR2 null keratinocytes formed only small skin tumors in orthotopic skin grafts to CXCR2 intact hosts, whereas transformed wild-type keratinocytes produced large tumors. In vitro, CXCR2 was essential for CXCR2 ligand-stimulated migration of ras-transformed keratinocytes and for ligand activation of the extracellular signal-regulated kinase (ERK) and Akt pathways. Both migration and activation of ERK and Akt were restored by CXCR2 reconstitution of CXCR2 null keratinocytes. Thus, activation of CXCR2 on ras-transformed keratinocytes has both promigratory and protumorigenic functions. The up-regulation of CXCR2 ligands after initiation by oncogenic ras and promotion with TPA in the mouse skin model provides a mechanism to stimulate migration by both autocrine and paracrine pathways and contribute to tumor development.

The high-risk benign tumor: evidence from the two-stage skin cancer model and relevance for human cancer.

Benign tumors that form following chemical initiation and promotion in the mouse skin can be grouped into two classes. The majority of papillomas do not progress to squamous cell carcinoma (SCC), and these are designated as low-risk or terminally benign papillomas. In contrast, a much smaller group forms the true precursor to the SCC, and these have a significantly higher frequency and rate of malignant conversion than the bulk of low-risk papillomas. In standard two-stage carcinogenesis studies both tumor types are present, but grossly indistinguishable. Here we describe properties and potential origins of high-risk papillomas and discuss the relevance of this model for certain human cancers with defined premalignant states.

Methylation of the O6-methylguanine-DNA methyltransferase promoter suppresses expression in mouse skin tumors and varies with the tumor induction protocol.

Hypermethylation of CpG sites within the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene occurs frequently in human cancer, preventing both MGMT expression and repair of alkylation damage. To assess the role of MGMT in the development of mouse skin tumors induced by initiation-promotion protocols, methylation of the MGMT promoter was examined in tumor DNA using methylation-specific PCR. To determine whether MGMT promoter methylation was affected by the tumor induction protocol, tumors were initiated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein. Although the MGMT promoter was not methylated in normal skin, promoter methylation was found in 56 of 136 papillomas (41.2%) and in 19 of 37 squamous cell carcinomas (51.4%). When methylation of the MGMT promoter was compared in the 4 treatment groups, hypermethylation was found more frequently in tumors initiated by DMBA and promoted by mezerein, a protocol associated with a high frequency of malignant conversion. Methylation was found in some tumors as early as 5 weeks after initiation, but the methylation frequency increased with time. MGMT promoter methylation reduced MGMT expression as determined by immunohistochemistry. Although MGMT promoter methylation was not generally correlated with ras mutations, the frequency of MGMT methylation was higher in MNNG-initiated, mezerein-promoted papillomas with mutations in Ha-ras compared to papillomas with Ki-ras. Methylation of the MGMT promoter, associated with reduced MGMT expression, is found in nearly half of mouse skin tumors, but varies with both the tumor initiator and tumor promoter, and may be a key step in the progression from papillomas to carcinomas.

Strain-dependent differences in malignant conversion of mouse skin tumors is an inherent property of the epidermal keratinocyte.

The multistage evolution of squamous skin tumors induced by chemical or viral carcinogens on mice from different genetic backgrounds has been a valuable model to define low penetrance loci that determine cancer susceptibility or resistance. Susceptibility determinants are multigenic, stage-specific, dependent on the carcinogenesis protocol, and in the case of initiating events, intrinsic properties of keratinocytes. In this study we examined the malignant conversion frequency of keratinocytes derived from FVB/N, inbred SENCARA/Pt, BALB/c or C57BL/6 mouse strains that differ substantially in the frequency of progression from papilloma to carcinoma. Keratinocytes were cultured from newborn mice and tested in an in vitro malignant conversion assay induced by a chemical carcinogen or immortalized by infection with replication defective human papillomavirus type 16 (HPV-16) E6/E7 retroviruses and tested for malignancy by grafting immortalized cell lines to nude mice. In vitro, FVB/N keratinocytes were 10-fold more sensitive to chemically induced malignant conversion than keratinocytes from other strains, consistent with the known sensitivity of this strain to pre-malignant progression in vivo. The E6/E7 genes induced immortalization of keratinocytes from FVB/N, SENCARA/Pt or C57BL/6 mice more efficiently than BALB/c, and HPV-16-immortalized FVB/N keratinocytes formed tumors more frequently (64%) than SENCARA/Pt (31%) BALB/c (1.9%) or C57BL/6 (2.5%). Furthermore, 78% of the tumors formed by FVB/N keratinocytes progressed to squamous carcinomas compared with 46% for SENCARA/Pt-derived cells and <3% for the others. In F(1) offspring of crosses from SENCARA/Pt and FVB/N mice, both the papilloma incidence and frequency of malignant conversion reflected the SENCARA/Pt parent indicating that predisposition to pre-malignant progression is not a dominant characteristic. This predisposition is an intrinsic property of the target keratinocytes and as such should be amenable to further study in isolated cells.

RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties.

Tripartite motif protein 32, Trim32, mRNA and protein expression was elevated in independently transformed and tumorigenic keratinocytes of a mouse epidermal carcinogenesis model, in ultraviolet B (UVB)-induced squamous cell carcinomas (SCC), and in approximately 20-25% of chemically induced mouse papillomas and human head and neck SCCs. This suggests that elevated Trim32 expression occurs frequently in experimental epidermal carcinogenesis and is relevant to human cancer. Transduced Trim32 increased colony number in an epidermal in vitro transformation assay and epidermal thickening in vivo when skin-grafted to athymic nu/nu mice. These effects were not associated with proliferation and were not sufficient for tumorigenesis, even with 12-O-tetradecanoylphorbol-13-acetate treatment or defects in the tumor suppressor p53. However, transduced Trim32 inhibited the synergistic effect of tumor necrosis factor alpha (TNFalpha) on UVB-induced apoptosis of keratinocytes in vitro and the apoptotic response of keratinocyte grafts exposed to UVB-light in vivo. Consistent with its RING domain, Trim32 exhibited characteristics of E3-ubiquitin ligases, including being ubiquitylated itself and interacting with ubiquitylated proteins, with increases in these properties following treatment of cultured keratinocytes with TNFalpha/UVB. Interestingly, missense point mutation of human TRIM32 has been reported in Limb-Girdle Muscular Dystrophy Type 2H, an autosomal recessive disease. We propose a model in which Trim32 activities as an E3-ubiquitin ligase favor initiated cell survival in carcinogenesis by blocking UVB-induced TNFalpha apoptotic signaling.

New amide-bearing benzolactam-based protein kinase C modulators induce enhanced secretion of the amyloid precursor protein metabolite sAPPalpha.

Protein kinase C (PKC) is known to participate in the processing of the amyloid precursor protein (APP). Abnormal processing of APP through the action of the beta- and gamma-secretases leads to the production of the 39-43 amino acid Abeta fragment, which is neurotoxic and which is believed to play an important role in the etiology of Alzheimer's disease. PKC activation enhances alpha-secretase activity, which results in a decrease of the amyloidogenic products of beta-secretase. In this article, we describe the synthesis of 10 new benzolactam V8 based PKC activators having side chains of varied saturation and lipophilicity linked to the aromatic ring through an amide group. The K(i) values measured for the inhibition of phorbol ester binding to PKCalpha are in the nanomolar range and show some correlation with their lipophilicity. Compounds 5g and 5h show the best binding affinity among the 10 benzolactams that were synthesized. By use of a cell line derived from an AD patient, significant enhancement of sAPPalpha secretion was achieved at 1 microM concentration for most of the compounds studied and at 0.1 microM for compounds 5e and 5f. At 1 microM the enhancement of sAPPalpha secretion for compounds 5c-h is higher than that observed for the control compound 8-(1-decynyl)benzolactam (BL). Of interest is the absence of activity found for the highly lipophilic ligand 5i, which has a K(i) of 11 nM. On the other hand, its saturated counterpart 5j, which possesses a comparable K(i) and ClogP, retains activity in the secretase assay. In the hyperplasia studies, 5f showed a modest response at 100 microg and 5e at 300 microg, suggesting that 5f was approximately 30-fold less potent than the PKC activator mezerein and 100-fold less potent than TPA. 5e was approximately 3-fold less active than 5f. On the basis of the effect of unsaturation for other potent PKC ligands, we would predict that 5e would retain biological activity in most assays but would show a marked loss of tumor-promoting activity. Compound 5e thus becomes a viable candidate compound in the search for Alzheimer's therapeutics capable of modulating amyloid processing.