It has been shown that UVB irradiation induces expression of COX-2 and up-regulation of COX-2 plays a functional role in UVB tumor promotion. In this study, we examined the cis-elements in the human COX-2 promoter that may be responsible for the UVB induction of COX-2. Analyses with the COX-2 promoter region revealed that the cyclic AMP responsive element near the TATA box was essential for both basal and UVB induced COX-2 expression. This was further supported by studies using a dominant negative mutant of CREB, which strongly inhibited the activity of COX-2 promoter. Electrophoretic mobility shift assays indicated that CREB and ATF-1 were the major proteins binding to the COX-2 CRE. CREB and ATF-1 were phosphorylated upon UVB treatment, and SB202190, a p38 MAPK inhibitor, decreased the phosphorylation of CREB/ATF-1 and suppressed COX-2 promoter activity. In contrast, treatment with forskolin, an activator of adenylyl cyclase, led to phosphorylation of CREB and ATF-1 and activation of COX-2 promoter. Finally, enhanced binding of phospho-CREB/ATF-1 to the COX-2 CRE was observed after UVB induction. Thus, one signaling pathway for UVB induction of human COX-2 involves activation of p38, subsequent phosphorylation of CREB/ATF-1, and activation of the COX-2 CRE through enhanced binding of phosphorylated CREB/ ATF-1. Oncogene (2001) 20, 5164 ± 5172.

Twenty-five surgical specimens of malignant human prostate, 3 lymph nodes with metastatic prostate carcinoma, 11 normal human prostates, as well as 3 human prostate cell lines (DU-145, PC3 and LNCaP) were examined for the expression of the human matrix metalloproteinase-7 gene (MMP-7) from the human collagenase family (originally called PUMP-1 for putative metalloproteinase-1) [Quantin et al. (1989) Biochemistry 28:5327-5334; Muller et al. (1988) Biochem J 253:187-192; Matrisian and Bowden (1990) Semin Cancer Biol 1:107-115]. Northern blots were prepared using total RNA extracted from 18 prostate adenocarcinomas, 2 lymph nodes with metastatic prostate carcinoma and 11 normal human prostates. When the northern blots were hybridized with a 32P-labeled MMP-7 cDNA probe, a 1.2-kb mRNA was detected in 14 out of 18 prostate adenocarcinomas, 1 out of 2 metastatic lymph nodes, and 3 out of 11 normal prostates. The 3 human prostate cell lines did not show any evidence of the MMP-7 transcript. In situ hybridization was conducted to localize the MMP-7 mRNA to individual cells using a 35S-labeled MMP-7 cRNA. In situ hybridization was carried out on snap-frozen tissue sections of 7 prostate adenocarcinomas and 3 lymph nodes containing metastatic prostate adenocarcinoma using the same tissues previously probed by northern analysis as well as new samples. In situ hybridization revealed that the MMP-7 gene was expressed in the epithelial cells of primary prostate adenocarcinoma as well as in invasive and metastatic cells. MMP-7 expression was also seen focally in some dysplastic glands but not in stroma. Additional northern blot analysis was performed using probes to human type-IV collagenase, type-I collagenase and stromelysin I in human prostate adenocarcinoma as well as normal prostate tissue. Our results indicated that 6 out of 10 adenocarcinoma samples and none of the 4 normal samples were positive for type-IV collagenase transcripts. Tissue samples were also examined for the expression of type-I collagenase (9 adenocarcinomas and 4 normal) and stromelysin I (13 adenocarcinomas) by northern analysis. None of the tissues was found to express the transcripts of interest at detectable levels. These data suggest that certain metalloproteinases are present in prostatic adenocarcinoma and may play a role in invasion and metastasis.

The mouse epidermal cell line 308 contains an activated Ha-ras gene and forms benign papillomas when transplanted to the skin of athymic nude mice. A radiation-associated malignant variant of this cell line, 308-10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in malignant conversion of 308-10Gy5, we assessed the activator protein-1 (AP-1) binding and transactivating ability of 308 and 308-10Gy5. In nuclear protein extracts of 308, AP-1 sequence-specific binding to an oligonucleotide containing a single high-affinity AP-1 binding site was induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, as determined by gel shift analysis. Nuclear extracts of 308-10Gy5 bound to the AP-1 oligonucleotide without treatment with tumor promoters. Not only was sequence-specific AP-1 DNA binding constitutively active in malignant versus benign tumor cells, but so was transactivation of a unique AP-1-responsive chloramphenicol acetyltransferase reporter construct, pTiCTaK. Constitutive transactivation of this AP-1-responsive reporter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady-state transcript levels of the tumor-associated AP-1-responsive genes stromelysin, urokinase-type plasminogen activator, c-jun, and c-fos were higher in malignant 308-10Gy5 cells than in benign 308 cells. These results suggest that acquisition of constitutive AP-1 DNA binding and transactivation can result in sustained deregulation of gene expression. While malignant progression in keratinocytes is probably not due solely to the acquisition of constitutive cellular AP-1 activity, the effect of deregulated expression of AP-1-regulated genes, especially basement membrane-degrading enzymes, may be functionally related to malignant conversion.

We have reported that transin RNA, a 1.9-kb RNA coding for a novel, secreted proteinase, was overexpressed during the progression of benign mouse skin papillomas to malignant squamous cell carcinomas (SCCs) induced by a two-stage protocol (Proc Natl Acad Sci USA 83:9413, 1986). Recently a high degree of similarity has been demonstrated between rabbit stromelysin, a secreted metalloproteinase that degrades proteoglycans found in the basement membrane and the amino acid sequence predicted in rat transin cDNA. DNA sequencing of a mouse cDNA isolated from an SCC (initiated by 7,12-dimethylbenz[a]anthracene [DMBA] and promoted by 12-O-tetradecanoylphorbol-13-acetate [TPA]) showed greater than 85% nucleotide similarity and 90% amino acid similarity to the rat transin-1 cDNA nucleotide and predicted amino acid sequences. Using this mouse transin cDNA clone as a probe (labeled with 32P) we found enhanced levels of transin mRNA transcripts in SCCs induced by a protocol giving rise to metastatic tumors (repeated N-methyl-N-nitroso-N'-nitroguanidine [MNNG] treatments) compared with the level found in SCCs induced by a protocol that had a lower probability of giving rise to metastatic tumors (MNNG initiation followed by TPA promotion). A study of primary SCCs and metastatic lesions induced by repeated benzo[a]pyrene treatment showed that the levels of transin mRNA transcripts were reduced in the metastatic lesions in comparison to the primary tumors. Southern analysis of the DNA isolated from epidermis, papillomas, and SCCs indicated that neither transin gene amplification nor rearrangement accounted for increased levels of the transin mRNA transcripts. These data suggest a role for enhanced levels of transin production in the invasion and metastasis of chemically induced SCCs.

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