Normal wound healing is characterized by an initial inflammatory response followed by reformation of the epithelial barrier and ECM deposition. Ideally, inflammation quickly resolves and tissue repair occurs in a regulated fashion. In contrast, we have found that wound repair in skin with elevated epidermal polyamine biosynthesis exhibits exaggerated epidermal hyperproliferation and persistent inflammation leading to the development of benign tumors. This wound-induced tumor formation was not observed in normal littermate mice and was dependent on ODC activity and polyamine biosynthesis since DFMO treatment normalizes the wound response and prevents tumor formation in transgenic mice. Remarkably, tumors developed in these animals in the absence of chemical carcinogens or the initiation of genetic lesions but were dependent on inflammatory cell recruitment resulting from the wounding and the elevated epidermal ODC activity.
Previously, we reported that elevated epidermal ODC activity in both K6/ODC and ODCER transgenic skin increases the proliferation index in the basal layer of non-wounded epidermis but does not stimulate inflammation nor result in epidermal hyperplasia or tumor formation (11
). Here, we show that wounding induces exaggerated epidermal hyperplasia and tumor growth in both K6/ODC and ODCER transgenic mice. Re-epithelialization following wounding involves recruitment of stem cells from the bulge area of the remaining hair follicles (37
). Because ODC expression was targeted to the epidermal compartment, we chose to abrade ODC transgenic mouse skin which only removes the epidermal layer. Abrasion precipitates a robust predictable epidermal hyperplastic response with relative absence of granulation tissue. In contrast, full thickness wounds result in both epidermal hyperplasia and extensive proliferation of the underlying connective tissue. Abrasion triggered a prolonged epidermal hyperplastic response in both K6/ODC and ODCER transgenic mice. Unlike the early hyperproliferative overshoot in K6/ODC abraded skin which was marked by hyperplasia of cells lining follicular-derived cysts in the dermis, the early wound healing response in inducible ODCER skin was similar to that in normal littermate skin at 7 days following abrasion. The proliferation rate in newly regenerated epidermis was similarly elevated in K6/ODC, ODCER and normal mice at 1 week following abrasion. However, papillomatous growths and increasing epidermal hyperplasia developed in both K6/ODC and ODCER transgenic skin by 14–21 days following abrasion when both the epidermal proliferation index and hyperplasia had significantly regressed in normal littermate skin. The epidermal proliferation rate remained significantly elevated in both K6/ODC and ODCER transgenic skin at 14 days following abrasion compared with that in normal littermates. Since the wound-induced tumor growth in K6/ODC and ODCER transgenic mice slowly regressed, the majority of wound-induced tumors in transgenic mice were most probably low risk, terminally benign papillomas (38
). Additional studies are needed to determine whether a small subgroup of these tumors can progress to a malignant phenotype.
Consistent with the exaggerated epidermal hyperplasia in abraded transgenic skin, Akt and mTOR signaling remains activated in ODCER and K6/ODC transgenic mice compared with that in normal littermates. In addition, polyamines are known to bind to RNA (39
) and modulate both translation initiation (40
) and elongation (42
). Specifically spermidine is required for the formation of hypusine which is incorporated in the posttranslational modification and activation of the translational elongation factor, eIF5A (33
). Up-regulation of these polyamine-modulated effectors of protein synthesis most probably plays an important role in sustaining the high proliferation rate and epidermal hyperplasia in wounded ODC transgenic skin. Because treatment with rapamycin did not reverse the exaggerated epidermal hyperplasia in abraded ODCER transgenic skin, it is probably that polyamine-stimulation of protein synthesis via activation of mTor signaling is not sufficient to sustain epidermal hyperplasia and wound-induced tumors in ODC transgenic skin. However, treatment with an anti-inflammatory agent dramatically reduced the epidermal hyperplasia in wounded ODCER skin without significantly reducing the high proliferation rate in the basal epidermal layer. Furthermore, inhibition of ODC activity with DFMO only normalized the wound healing response when given from the time of abrasion but not when initiated a few days after abrasion. DFMO suppressed not only the epidermal proliferation rate but also the early infiltration of neutrophils in newly abraded skin. Thus, although elevated polyamine levels stimulate epidermal proliferation both in non-wounded (11
) and wounded skin, recruitment of inflammatory cells is necessary to sustain epidermal hyperplasia and wound-induced tumors.
The persistent infiltration of inflammatory cells in abraded ODC transgenic skin is also accompanied with a significant increase in wound MCP-1 production. MCP-1 levels strongly influence the infiltration of macrophages into wounds as demonstrated by the strong influx of macrophages following topical application of recombinant MCP-1 to wounds (43
). Interestingly, mice that are deficient in MCP-1 exhibit defective wound repair but with no change in the number of wound macrophages, implicating a role for MCP-1 in macrophage activation and in promoting epithelial and vascular growth (44
). It is possible that elevated levels of MCP-1 alters the effector state of macrophages and other inflammatory cells, thus contributing to the dramatic hyperplastic wound response and wound-induced tumor formation. For instance, macrophage polarization from an M1 proinflammatory phenotype to an M2 pro-growth phenotype (45
) may be induced in a microenvironment with elevated epidermal levels of polyamines. Besides the beneficial role of inflammatory cells in killing invading bacteria in the wound bed, a persistant inflammatory microenvironment found in a chronic wound can also be a risk factor for malignant transformation (36
). Our data suggest that elevated polyamine biosynthesis contributes to ‘a wound that never heals’ phenotype that Dvorak (16
) has likened to a tumor.
Chronic wounds are characterized by a persistent inflammatory response at the wound site, accompanied by unbalanced proteolytic activity and a highly pro-oxidant microenvironment (36
). Infiltrating leukocytes, including neutrophils and macrophages, are a rich source of various reactive oxygen species that are released into the wound. In addition, the polyamine catabolic enzyme spermine oxidase, that generates both reactive oxygen species and cytotoxic aldehydes, is induced in ODC overexpressing keratinocytes (47
). Since we found that the polyamine catabolic enzyme spermine oxidase is upregulated in the hyperplastic epidermis of abraded ODCER transgenic mice, it is possible that increased polyamine metabolism may contribute to the generation of a more pro-oxidant wound environment and the resulting prolonged hyperplasia and tumor growth in wounded ODCER mice. However, we have found that treatment with neither the reactive oxygen species scavenger, N
-acetyl cysteine (48
) nor with MDL-72527, an inhibitor of spermine oxidase activity (49
), prevented the wound-induced tumor formation in ODCER transgenic mice (data not shown). Our data suggest that increased epidermal polyamine biosynthesis exacerbates the wound healing process via multiple mechanisms that stimulate both epithelial proliferation and recruitment of inflammatory cells.
This study shows that wounding cooperates with increased epidermal ODC activity in non-dividing suprabasal epidermal cells to initiate tumor formation. Arwert et al.
) also recently reported benign skin tumor growth following wounding in transgenic mice in which expression of activated mitogen-activated protein kinase kinase 1 is directed to suprabasal, non-dividing epidermal cells. The wound-initiated tumor formation in their transgenic mouse model was dependent on inflammatory cells including γδ T cells and macrophages (51
). A similar wound healing phenotype is found in ODCER transgenic skin which is characterized by epidermal hyperproliferation with prolonged activation of translation-associated proteins, a prolonged infiltration of inflammatory cells, activation of fibroblasts and increased vascularization, all of which provide a growth factor and cytokine-enriched stroma that can both support and promote tumorigenesis. We propose that the ODCER transgenic mouse model of wound healing provides a novel animal model to study the role of chronic inflammation in malignant transformation. In addition to stimulating proliferation of newly regenerated epidermal cells, this study highlights the far-reaching epigenetic effects that elevated epidermal polyamines impose on the dermal microenvironment to activate underlying stromal cells, enhance recruitment of inflammatory cells and skew a normal wound healing response into pathology involving tumor growth. Because these activating, modulatory effects of polyamines act as an accelerating switch in tissue remodeling events such as wound healing, inhibition of polyamine metabolic pathways is an important consideration in designing not only chemopreventive therapy but also adjunct chemotherapy in the treatment of skin cancer.