Search

To define a functional role for the endosomal/lysosomal cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Ctsl deficiency in addition to epithelial expression of the human papillomavirus type 16 oncogenes (human cytokeratin 14 (K14)-HPV16). We found enhanced tumor progression and metastasis in the absence of Ctsl. As tumor progression in K14-HPV16 mice is dependent on inflammation and angiogenesis, we examined immune cell infiltration and vascularization without finding any effect of the Ctsl genotype. In contrast, keratinocyte-specific transgenic expression of cathepsin V, the human orthologue of mouse Ctsl, in otherwise Ctsl-deficient K14-HPV16 mice restored the phenotype observed in the control HPV16 skin. To better understand this phenotype at the molecular level, we measured several oncogenic signal transduction pathways in primary keratinocytes on stimulation with keratinocyte-conditioned cell culture medium. We found increased activation of protein kinase B/Akt and mitogen-activated protein kinase pathways in protease-deficient cells, especially if treated with media conditioned by Ctsl-deficient keratinocytes. Similarly, the level of active GTP-Ras was increased in Ctsl-deficient epidermis. We conclude that Ctsl is critical for the termination of growth factor signaling in the endosomal/lysosomal compartment of keratinocytes and, therefore, functions as an anti-tumor protease.

The matrix metalloproteinase MMP-9/gelatinase B is upregulated in angiogenic dysplasias and invasive cancers of the epidermis in a mouse model of multi-stage tumorigenesis elicited by HPV16 oncogenes. Transgenic mice lacking MMP-9 show reduced keratinocyte hyperproliferation at all neoplastic stages and a decreased incidence of invasive tumors. Yet those carcinomas that do arise in the absence of MMP-9 exhibit a greater loss of keratinocyte differentiation, indicative of a more aggressive and higher grade tumor. Notably, MMP-9 is predominantly expressed in neutrophils, macrophages, and mast cells, rather than in oncogene-positive neoplastic cells. Chimeric mice expressing MMP-9 only in cells of hematopoietic origin, produced by bone marrow transplantation, reconstitute the MMP-9-dependent contributions to squamous carcinogenesis. Thus, inflammatory cells can be coconspirators in carcinogenesis.

Expression of HPV16 early region genes in basal keratinocytes of transgenic mice elicits a multistage pathway to squamous carcinoma. We report that infiltration by mast cells and activation of the matrix metalloproteinase MMP-9/gelatinase B coincides with the angiogenic switch in premalignant lesions. Mast cells infiltrate hyperplasias, dysplasias, and invasive fronts of carcinomas, but not the core of solid tumors, where they degranulate in close apposition to capillaries and epithelial basement membranes, releasing mast-cell-specific serine proteases MCP-4 (chymase) and MCP-6 (tryptase). MCP-6 is shown to be a mitogen for dermal fibroblasts that proliferate in the reactive stroma, whereas MCP-4 can activate progelatinase B and induce hyperplastic skin to become angiogenic in an in vitro bioassay. Notably, premalignant angiogenesis is abated in a mast-cell-deficient (KITW/KITWWv) HPV16 transgenic mouse. The data indicate that neoplastic progression in this model involves exploitation of an inflammatory response to tissue abnormality. Thus, regulation of angiogenesis during squamous carcinogenesis is biphasic: In hyperplasias, dysplasias, and invading cancer fronts, inflammatory mast cells are conscripted to reorganize stromal architecture and hyperactivate angiogenesis; within the cancer core, upregulation of angiogenesis factors in tumor cells apparently renders them self-sufficient at sustaining neovascularization.

Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.