Boris Shorning

Actinic keratoses (AKs) represent the precursor skin lesions in the pathogenesis of up to 87% of cutaneous squamous cell carcinomas (cSCCs). However, current treatment of AK lesions, at best, lead to only a 25% reduction in cSCC and relies on therapies that target abnormal keratinocytes using topical chemotherapy, immune stimulants or surgical resection. We have previously shown that 44% of AK tissue sample histologies contain koilocytes, wherein genotyping universally identified human papillomavirus 8 (HPV8). To determine the role of HPV8 in the pathogenesis of AKs, we used the K14–HPV8 complete early region (HPV8tg) to generate triple cross-lineage tracing transgenic mice: HPV8:K5-CreERT2:R26R-LoxP-flanked-Brainbow2.1 (Confetti reporter); HPV8:Lrig1-CreERT2:R26R-LoxP-flanked-Brainbow2.1; and HPV8:K15-PGR:R26R-LoxP-flanked-Brainbow2.1. Upon Cre activation, progeny clones exhibit one of four fluorescent colours. Compared to wild-type mice, all HPV8tg mice exhibited an expansion of keratinocytes in the hair follicle infundibulum (77.09 mm³ and 253.43 ± 54.15 mm³) and overlying epidermis. Clones from the Lrig1, but not K15, keratinocyte stem cell population were responsible for keratinocyte expansion of both the hair follicle infundibulum and overlying epidermis. Compared with wild-type mice, HPV8tg mice exhibited greater colony-forming efficiency (1.75 ± 0.69% vs. 6.27 ± 2.41%; n = 3), and, consistent with keratinocyte stem cell expansion, ΔNp63 expression was increased 4.14 ± 2.10-fold (P < 0.05, n = 5). Flow-sorted epidermal clones from wild-type and HPV8tg mice were examined by RNA sequencing (n = 5). Warthin–Starry staining showed that the expanded population had no melanin ‘cap’ in our mouse models and human AK samples. In summary, expression of HPV8 early-region genes is associated with AK, wherein there is an expansion of the Lrig1, but not K15, keratinocyte stem cell population that results in epidermal thickening reminiscent of human AK pathology. These findings suggest that HPV8 results in translocation of hair follicle keratinocyte stem cells into the interfollicular epidermis without melanin production, making these cells susceptible to ultraviolet light transformation.

Model of possible effects of P1597L mutation

Metastatic deposit in Ptenfl/flKrasG12VPLXNB1WT mouse

Proliferation and ROCK activation in Ptenfl/flKrasG12V PlxnB1-/- and Ptenfl/flp53fl/fl PlxnB1-/- mice

Metastatic prostate cancer is essentially incurable and is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer.We show here that prostate epithelial cell–specific expression of a mutant form of Plexin-B1 (P1597L) which was identified in metastatic deposits in patients with prostate cancer, significantly increases metastasis, in particular metastasis to distant sites, in two transgenic mouse models of prostate cancer (PbCre+Ptenfl/flKrasG12Vand PbCre+Ptenfl/flp53fl/fl). In contrast, prostate epithelial cell–specific expression of wild-type (WT) Plexin-B1 in PbCre+Ptenfl/flKrasG12V mice significantly decreases metastasis, showing that a single clinically relevant Pro1597Leu amino-acid change converts Plexin-B1 from a metastasis-suppressor to a metastasis-promoter. Furthermore, PLXNB1P1597L significantly increased invasion of tumor cells into the prostate stroma, while PLXNB1WTreduced invasion, suggesting that Plexin-B1 has a role in the initial stages of metastasis. Deletion of RhoA/C or PDZRhoGEF in Ptenfl/flKrasG12VPLXNB1P1597L mice suppressed metastasis, implicating the Rho/ROCK pathway in this phenotypic switch. Germline deletion of Plexin-B1, to model anti-Plexin-B1 therapy, significantly decreased invasion and metastasis in both models.Our results demonstrate that Plexin-B1 plays a complex yet significant role in metastasis in mouse models of prostate cancer and is a potential therapeutic target to block the lethal spread of the disease.Significance:Few therapeutic targets have been identified specifically for preventing locally invasive/oligometastatic prostate cancer from becoming more widely disseminated. Our findings suggest Plexin-B1 signaling, particularly from the clinically relevant P1597L mutant, is such a target.

Metastatic deposits in Ptenfl/flKrasG12VPLXNB1P1597L mice

Metastatic deposits in Ptenfl/flp53fl/fl mice

Summary of age of mice and weight of primary tumours