Actinic keratoses (AKs) are lesions of epidermal keratinocyte dysplasia and are precursors for invasive cutaneous squamous cell carcinoma (cSCC). Identifying the specific genomic alterations driving the progression from normal skin to skin with AK to skin with invasive cSCC is challenging because of the massive UVR-induced mutational burden characteristic at all stages of this progression. In this study, we report the largest AK whole-exome sequencing study to date and perform a mutational signature and candidate driver gene analysis on these lesions. We demonstrate in 37 AKs from both immunosuppressed and immunocompetent patients that there are significant similarities between AKs and cSCC in terms of mutational burden, copy number alterations, mutational signatures, and patterns of driver gene mutations. We identify 44 significantly mutated AK driver genes and confirm that these genes are similarly altered in cSCC. We identify azathioprine mutational signature in all AKs from patients exposed to the drug, providing further evidence for its role in keratinocyte carcinogenesis. cSCCs differ from AKs in having higher levels of intrasample heterogeneity. Alterations in signaling pathways also differ, with immune-related signaling and TGFβ signaling significantly more mutated in cSCC. Integrating our findings with independent gene expression datasets confirms that dysregulated TGFβ signaling may represent an important event in AK‒cSCC progression.

β-Human papillomaviruses (HPVs) cause near ubiquitous latent skin infection within long-lived hair follicle (HF) keratinocyte stem cells. In patients with epidermodysplasia verruciformis, β-HPV viral replication is associated with skin keratosis and cutaneous squamous cell carcinoma. To determine the role of HF keratinocyte stem cells in β-HPV-induced skin carcinogenesis, we utilized a transgenic mouse model in which the keratin 14 promoter drives expression of the entire HPV8 early region (HPV8tg). HPV8tg mice developed thicker skin in comparison with wild-type littermates consistent with a hyperproliferative epidermis. HF keratinocyte proliferation was evident within the Lrig1+ keratinocyte stem cell population (69 vs. 55%, P < 0.01, n = 7), and not in the CD34+, LGR5+, and LGR6+ keratinocyte stem cell populations. This was associated with a 2.8-fold expansion in Lrig1+ keratinocytes and 3.8-fold increased colony-forming efficiency. Consistent with this, we observed nuclear p63 expression throughout this population and the HF infundibulum and adjoining interfollicular epidermis, associated with a switch from p63 transcriptional activation isoforms to ΔNp63 isoforms in HPV8tg skin. Epidermodysplasia verruciformis keratosis and in some cases actinic keratoses demonstrated similar histology associated with β-HPV reactivation and nuclear p63 expression within the HF infundibulum and perifollicular epidermis. These findings would suggest that β-HPV field cancerization arises from the HF junctional zone and predispose to squamous cell carcinoma.

The precise mechanisms governing invasion at the leading edge of squamous cell carcinoma (SCC) and its subsequent metastasis are not fully understood. We aimed to define the cancer-related molecular changes that distinguish noninvasive tumor from invasive SCC. To this end, we combined laser capture microdissection with complementary DNA (cDNA) microarray analysis. We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared with normal epidermis. There were 383 upregulated and 354 downregulated genes in the "invasion set." SCC invasion was characterized by aberrant expression of various proteolytic molecules. We noted increased expression of MMP7 and IL-24 in invasive SCC. IL-24 induced the expression of matrix metallopeptidase 7 (MMP7) in SCC cells in culture. In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture. These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, although IL-24 has been suggested to have antitumor growth effects in other cancer types. Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer.