Furin is a serine protease that is frequently overexpressed in several human cancer cell lines and malignancies [24,26,27,36,42,43
] including several murine cell lines derived from skin tumors induced by chemical carcinogenesis [44
]. Its activity results in proteolytic cleavage of substrates leading to activation of many cancer-related proteins including important growth factors and receptors such as IGF-1 and its receptor IGF-1R, TGF-β, etc. [8,30,45–47
To evaluate the in vivo
effects of furin on skin biology and tumorigenesis, we developed two K5-Furin mouse lines (F47 and F49). Real-time PCR and Western analyses showed that F47 mice expressed approximately twice as much furin as F49 mice at the protein level. IHC analysis of both lines revealed increased number of basal keratinocytes expressing furin. The enhanced furin expression was reflected in higher levels of activated IGF-1R as demonstrated by Western blot analysis. This increase was more obvious and significant in F47 than in F49 mice. Similarly, the basal keratinocyte proliferation rate of the epidermis in F47 mice was significantly elevated over the WT and F49 counterparts. Interestingly, this difference was more prominent and significant in the infundibulum or intrafollicular segment of the epidermis than in the interfollicular epidermis. This difference is even more noteworthy if one takes into account several reports pointing to the hair follicle and, in particular, to the suprasebaceous component of this epidermal appendix as the origin of nonmelanoma skin neoplasia [48–50
In a previous report, we found that another PC, PACE4, had a pivotal role in enhancing mouse skin tumor development and invasion [35
]. Because PACE4 is localized extracellularly, it has easy access to intercellular and matrix-associated substrates such as metalloproteases and is able to enhance tumor cell invasion. Furin, however, might have a more prominent role in activating intracellular cancer-related substrates that may influence other cellular processes such as proliferation. This possible divergence in function is supported by our results showing that keratinocytes derived from untreated transgenic mice expressing furin in the epidermal basal layer as well as tumor cells derived from transgenic animals were able to process IGF-1R and TGF-β. As a consequence of increased growth factor processing, transgenic epidermis showed a higher proliferative rate than control WT epidermal cells in vivo
. Conversely, we were unable to detect differences in processing of MT1-MMP and MT2-MMP in WT and transgenic epidermal cells derived from the two animal lines F47 and F49 (not shown). In a previous report [35
], we demonstrated that K5-PACE4 keratinocytes were able to process membrane-type MMPs and have an increased invasive ability, suggesting that furin and PACE4 may exert different functions, despite their similar enzyme properties.
To demonstrate a causal relationship between furin expression and increased tumorigenesis in vivo, we studied furin-overexpressing transgenic and WT mice treated with a two-stage skin carcinogenesis protocol. During the entire experimental period after the appearance of benign tumors or papillomas, the mean number of papillomas/mouse was higher in both transgenic mouse lines than in WT mice. More importantly, the mean number of SCC/mouse showed a similar tendency, in both lines at 30 weeks of treatment with the two-stage carcinogenesis protocol; the number of carcinomas per animal was higher than in WT mice, although this difference was most marked in F47 mice. Moreover, the incidence of malignant tumors, which is defined as the percentage of animals harboring carcinomas, was consistently higher in transgenic mice. In addition, tumor multiplicity and tumor volume were remarkably higher in transgenic mice when compared with WT mice, probably reflecting the increased proliferative rate evidenced in short-term in vivo experiments as well as by the increased processing of IGF-1R and TGF-β seen in carcinomas derived from transgenic mice.
Metastases in axial lymph nodes and lung were also higher in number in transgenic mice than in WT mice. Because of the low numbers of animals bearing metastases (mice had to be euthanized at 30 weeks because of the large and numerous skin primary tumors), the results were not statistically significant.
To assess a possible interference of the transgenic expression of furin in epidermal keratinocytes, we conducted an evaluation of the RNA expression of six other PCs in normal keratinocytes and SCCs derived from WT, F47, and F49 mice. The quantitative real-time PCR results showed that enforced furin expression had no or had minimal effect on the levels of PACE4, PC5, and PC7, whereas PC1 and PC4 were undetectable in both normal keratinocytes and SCC cells. PC2 was detected at higher levels in transgenic keratinocytes than in their WT counterparts. This effect may be due to a higher number or an enhanced survival of Merkel cells in the short-term cultures of transgenic epidermal cells. Merkel cells are known to express high levels of PC2 [51
]. Because this difference was not seen in the tumor cells where PC2 was undetectable in WT, F47, and F49 SCCs, it is doubtful that transgenic furin could have an enhancing effect on PC2 transcript expression during tumor development.
The data indicate that increased furin expression enhanced tumor development and growth without significantly affecting the expression of other endogenous PCs. It is noteworthy that the higher prevalence of SCCs in the transgenic line F47 versus line F49 correlated well with the relatively higher expression of furin in basal keratinocytes of line F47 as well with its increased baseline keratinocyte proliferation rate and also with a higher expression of furin in the SCCs of line F47 when compared with their counterparts in line F49, suggesting that there might be a dose-response type effect between the transgene level of expression and the susceptibility to skin chemical carcinogenesis.
Taken together, furin expression targeted in vivo
to the epidermis resulted in increased epidermal proliferation as well as increased cancer-related substrate activation that had a protumorigenic effect that was demonstrated by an increased susceptibility to carcinogen-induced skin tumors as exemplified by an enhanced SCC multiplicity and tumor volume. Because furin expression and activity are enhanced in lung and head and neck cancers [24,26,27,36,42,43
], these experimental data further support the possible use of furin as a target in human cancer treatment.